SYNTHÈSE

En développant un nouveau protocole SELEX basé sur l'oxydation au periodate, le but de notre travail est de découvrir de nouveau ribozymes d'une taille de l'ordre d'une vingtaine de nucléotides pouvant combiner la catalyse de l'activation des acides aminés et la transestérification. Bien que des molécules catalysant l'une ou l'autre des deux réactions ont été identifiées, aucun ribozyme n'existe à ce jour qui puisse utiliser des acides aminés libres et un cofacteur activant pour réaliser l'aminoacylation en 3' dans un même milieu réactionnel.

La sélection de molécules actives dans une approche SELEX exige la présence de régions constantes sur les deux extrémités des séquences pools aléatoires initiaux. Ces régions sont nécessaires pour l'amplification par PCR, mais elles imposent des contraintes importantes pour l'identification de ribozymes car elles peuvent complètement inhiber leur activité par interférence structurelle. Nous présentons un protocole optimisé qui minimise la taille de ces régions constantes. D'autre part, notre nouveau design est très spécifique pour la sélection d'ARN aminoacylés sur l'extrémité 3'. Ce protocole a été utilisé pour réaliser 6 à 7 cycles de sélection avec différents pools, et un enrichissement en séquences spécifiques a pu être mis en évidence. Bien que certains tests avec les pools sélectionnés aient révélé une activité possible, des essais avec des séquences spécifiques de ces pools n'ont pour l'instant pas pu confirmer l'activité catalytique recherchée.

Un protocole basé sur le même principe de sélection a été utilisé dans une étude parallèle pour identifier les ARN aminoacylés présents dans l'ARN total d'Escherichia Because it is required for translation, RNA aminoacylation plays an important role in the evolution from the late RNA world to the modern DNA and protein world, and is central to the genetic code. Several ribozymes catalyzing amino acid transfer from various activating groups have already been selected and characterized in the past two decades, documenting the possibility of tRNA aminoacylation in the absence of aminoacyl tRNA synthetase.

With a newly designed SELEX protocol based on periodate oxidation, the aim of our investigation is to uncover small ribozymes of the order of 20 nucleotides that could catalyze both amino acid activation and transesterification. Although molecules catalyzing either reaction have been identified, no existing ribozyme could use free amino acids and activating cofactor(s) as substrates for 3' esterification in a single reactional context.

The selection of active molecules in a SELEX procedure requires the presence of constant tracks on both ends of the sequences constituting the initial random pools.

These tracks are required for PCR amplification, but they impose significant burden to the identification of ribozymes because they can prevent any activity through structural inhibition. We present an optimized protocol that significantly minimizes the size of these constant tracks. At the same time, our newly design protocol is very specific for the selection of 3'-end aminoacylated RNA. Working with this protocol, we performed 6 to 7 cycles of selection with different pools, and observed an enrichment with specific sequences. Although some experiments performed with entire pools did reveal a possible activity, no activity could be so far confirmed with specific sequences.

A similar protocol was also applied in a parallel study to identify aminoacylated RNA from total RNA in Escherichia coli. In this other approach, our goal is to possibly identify new classes of aminoacylated RNA while using the deep sequencing technology. Using tRNA to validate our protocol, we realized that a standard RNAseq procedure could not work due to the presence of modified bases. We established a new method for bank preparation to identify any sequence aminoacylated at the 3' end.

Ultimately, this new approach will allow us to study the level of aminoacylation of any sequence present in total RNA. In the end, I would like to thank my parents, YAO Ruling and WANG Feng. During these four years far away from China, I feel sorry that I cannot be with them so often. Without their comprehensive and unconditional supports, it is impossible for me to finish my study.

I wish all the best to the colleagues from Institut de Biologie Inté grative de la Cellule (I2BC) and appreciate all the helps from them. straightforward to infer the composition or the metabolism of the primary form(s) of life. There is some kind of agreement that the original life rooted in the evolution of complex chemical mixtures (Figure 1) [START_REF] Grossenbacher | AMINO ACIDS, PEPTIDES, AND SPHERULES OBTAINED FROM "PRIMITIVE EARTH[END_REF][START_REF] Oro | Historical Understanding of Life's Beginnings[END_REF]. Biochemists have already proposed several hypotheses to explain how a "Spontaneous generation" could happen [START_REF] Balme | Development of Biology in Aristotle and Theophrastus: Theory of Spontaneous Generation[END_REF]) and how the organic compounds constituting the original chemical metabolism in the "Primordial soup" came about [START_REF] Shapiro | Origins: A skeptic's guide to the creation of life on earth[END_REF].

Figure 1. Chemical evolution has been undergoing several stages. In the first stage, simple organic substances, such as amino acids, nucleic acids, fatty acids, and sugar, start to form the primordial soup (Bailey 1938). Then the simple organic molecules begin to gathering together to yield larger macromolecules. As the protobiont evolved, it gained the ability of reproduction and delivering the genetic information (Morowitz et al. 1988). Finally, some of the polymers, such as RNA, DNA, peptide, are able to catalyze enzymatic reactions, construct the protocell and build the early life forms [START_REF] Chen | RNA Catalysis in Model Protocell Vesicles[END_REF].

During chemical evolution, it has been hypothesized that different molecules existing in the ancient ocean have all been through a natural selection based on their chemical properties [START_REF] Schwartz | Molecular Evolution towards the Origin of Living Systems on the Earth and elsewhere. IX und 278 S., 154 Abb., 24[END_REF]. RNA and DNA are not the only polymers to be evolutionary candidates.

Hypothetical biology building blocks, such as threose nucleic acid (TNA), peptide nucleic acid (PNA), glycerol-derived nucleic-acid analogue (GNA), (Figure 2) share a similar chemical structural profile and are able to form stable Watson-Crick base pairs just like RNA and DNA [START_REF] Joyce | The antiquity of RNA-based evolution[END_REF]. Furthermore, self-replication could also happen with certain peptides and small organic compounds without "Darwin evolution" [START_REF] Tjivikua | Self-replicating system[END_REF]; [START_REF] Lee | A self-replicating peptide[END_REF][START_REF] Murtas | Early self-reproduction, the emergence of division mechanisms in protocells[END_REF]. Thus, information storage and self-replication are not the specialty of nucleic acid only.

Nevertheless, RNA and DNA have some advantages over other elementary building blocks that prevailed. Nucleic acids are polymers made up of 4 different types of elementary units which can provide a high diversity of sequences, allow them to be replicated faithfully (compared to peptides), and provide the basis for Darwinian evolution [START_REF] Joyce | RNA evolution and the origins of life[END_REF]. Hypothetically, the original instability of individual RNA or DNA molecules and a low replication efficiency would make very long polymers impossible, and 50-100 bases have been estimated as an upper limit [START_REF] Eigen | The Hypercycle[END_REF]. However, at some point, an improved stability could promote the assemblage of larger molecules [START_REF] Yakhnin | A model for the origin of life through rearrangements among prebiotic phosphodiester polymers[END_REF]. Being enriched in electron donors, such as purine and pyrimidine groups, phosphate group and oxygen group, long RNA molecules may better position the ions binding location and form the initial enzymatic catalysis system [START_REF] Ralser | The RNA world and the origin of metabolic enzymes[END_REF].

Unlike DNA, single stranded RNA molecules can fold into extraordinary complicated secondary structures [START_REF] Uzman | Molecular biology of the cell[END_REF], although elementary folds (Figure 3) are common to the two polymers. An even more fundamental property of RNA is its large repertoire of catalytic activities that makes it far more functional than DNA. The first catalytic RNA was discovered in 1982. RNA enzymes, the so-called ribozymes, have changed our view on molecular biology [START_REF] Kruger | Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena[END_REF]. Right after the function of RNase P was established, more natural ribozymes were discovered in vivo. These ribozymes are like ancient fingerprints of the early RNA world and inspired scientists to explore the early form of life [START_REF] Baer | The recognition by RNase P of precursor tRNAs[END_REF]. The discovery of natural ribozymes has drawn the interest of scientists towards understanding their catalytic mechanisms. Meanwhile, in vitro selection was rapidly developing in laboratories and led to the discovery of various artificial ribozymes in past few decades [START_REF] Chen | Ribozyme catalysis of metabolism in the RNA world[END_REF]. It turns out that RNA molecules are far more functional than initially assumed.

More and more researchers now believe that RNA molecules are a major player during the evolution of the early life [START_REF] Pressman | The RNA World as a Model System to Study the Origin of Life[END_REF]. However, some fundamental issues such as the evolution of the genetic code are still a big puzzle. Also, how and why the catalysis of some chemical reactions possibly switched from RNA to protein and how the storage of genetic information switched from RNA to DNA is still debated.

The RNA world hypothesis

Since Walter coined the "RNA world" 30 years ago, the RNA world hypothesis has become a popular theory. This hypothesis stipulates that there are three stages in the evolution from the RNA world to the modern DNA/RNA and protein world: (I) RNA molecules were self-assembled based on their auto-catalytic activities and accumulated by self-replication, recombination and mutation. These processes made them explore new functions. By employing cofactors (ions, peptide, amino acid, etc.) [START_REF] Ae | Metal ions: supporting actors in the playbook of small ribozymes[END_REF], RNA molecules were able to develop an entire range of enzymatic activities (Figure 4); (II) RNA adapter molecules recruited from a pool of RNA molecules could bind different amino acids and polymerize them while using RNA templates: protein synthesis began. Co-evolution would promote the RNA/protein world establishment [START_REF] Copley | The origin of the RNA world: Co-evolution of genes and metabolism[END_REF]); (III) DNA molecules eventually replaced RNA as the template information holder [START_REF] Gilbert | The RNA world[END_REF]): the RNA/protein world switched to DNA/protein world.

Because of the irreversibility of evolution, the RNA world has disappeared billions of years ago and it is impossible to find physical traces of this world in the environment of early geological times. However, the RNA world hypothesis is still supported by some existing facts, such as the discovery of natural ribozymes, while the core structure of ribosome which is also based on RNA, and there are plenty RNA viruses [START_REF] Robertson | The Origins of the RNA World[END_REF][START_REF] Flores | Viroids: Survivors from the RNA World?[END_REF].

Moreover, in vitro ribozyme selection also showed that RNA molecules can catalyze various biochemical reactions. As described by the hypothesis, there are three critical functions that RNA molecules had to manage during the first stage: 1) self-replication (to undergo natural selection) [START_REF] Robertson | The Origins of the RNA World[END_REF]; 2) catalyzing various chemical reactions (to transform energy) (Morowitz et al. 1988);

and 3) aminoacylation (that linked the RNA world to the protein world) [START_REF] Yarus | Getting past the RNA world: the initial Darwinian ancestor[END_REF]).

In the very beginning, the RNA evolution started when it gained the ability to replicate, ligate, and/or perform splicing, so that a process equivalent to natural selection could begin (Figure 5). Former investigations in the 80s showed that single nucleotide can yield polyoligomers in the presence of divalent cation at alkaline pH, 0°C after a few days of incubation with a mineral catalyst [START_REF] Sleeper | The catalysis of nucleotide polymerization by compounds of divalent lead[END_REF][START_REF] Orgel | RNA catalysis and the origins of life[END_REF]). This indicates one way through which RNA could self-assemble. Even though polymerization likely happened extremely slowly and inefficiently, average size nucleotides could accumulate during long geological time scale. Furthermore, molecular cooperation among short RNA molecules would significantly increase the variability of polynucleotide chains through recombination, that might have provided favorable conditions to the evolution of larger macromolecules [START_REF] Higgs | The RNA World: molecular cooperation at the origins of life[END_REF]. Once the first template-directed replicating ribozyme came about, the growth of polynucleotide chains dramatically accelerated [START_REF] Ekland | RNA-catalysed RNA polymerization using nucleoside triphosphates[END_REF][START_REF] Martin | RNA Synthesis by in Vitro Selected Ribozymes for Recreating an RNA World[END_REF].

Complementing elongation, self-cleaving is an important way to increase variability during (1) Random activated monomers randomly self-assembled into functional and non-functional oligonucleotides (2), which could serve as templates (3) that guide the complementary strand synthesis (4). Inter-and intramolecular recombination allows more copies of RNA transcripts to yield (5) to promote the replication and evolution (6).

Being through the natural selection, RNAs could evolved to more sophisticated structures and functions (7), leading to the appearance of the first protocell and the early biocatalysts of the RNA world (8). Illustration from [START_REF] Hernandez | Chemical origins of life: Prebiotic RNA unstuck[END_REF].

In vitro selections achieved since the 90s showed that RNA can accelerate many chemical reactions, such as amide bond formation, aldol reaction, Claisen condensation, Michael addition, thiol ester formation, Diels -Alder reaction and redox reactions [START_REF] Chen | Ribozyme catalysis of metabolism in the RNA world[END_REF]). These functions do not tell how was the early metabolism. However, they indicate that RNA molecules are able to catalyze various reactions in addition to those associated with natural ribozymes [START_REF] Jä Schke | Evolution of DNA and RNA as catalysts for chemical reactions[END_REF]. Based on many in vitro experiments, scientists have proposed chemical models to explain ribozyme catalytic mechanisms, in which the phosphate-ribose skeleton is the scaffold and nucleobases mainly govern ligand binding and construct active core formation [START_REF] Scott | Ribozymes: Structure and mechanism in RNA catalysis[END_REF][START_REF] Derose | Two decades of RNA catalysis[END_REF][START_REF] Lönnberg | Chemical models for ribozyme action[END_REF]. Moreover, these investigations not only helped us understand the function of natural ribozymes, these ribozymes are now also used as tools in cellular research [START_REF] Cotten | Ribozyme mediated destruction of RNA in vivo[END_REF][START_REF] Ohkawa | Ribozymes: from mechanistic studies to applications in vivo[END_REF][START_REF] Vourekas | Insights into functional modulation of catalytic RNA activity[END_REF]. Discovering new ribozymes is a goal still pursued by many laboratories today, and will allow us to expand our knowledge about RNA catalysis and the evolution of life from the early RNA world.

When the RNA world reached the gate of stage (II), a major transition was about to occur, that led the system to catalyze protein polymerization using RNA as templates. For this transition to occur, some RNA (the early tRNA adaptor) must have had the ability to undergo 3' aminoacylation, either through their own action (auto-aminoacylation) or by the action of other RNA/peptide cofactors. In the modern RNA aminoacylation system, specific aminoacyl tRNA synthetase catalyze both amino acids activation and tRNA 2'/3'-end transesterification.

ATP is employed as the energy driver to allow substrates to pass the reaction barrier (Figure 6). While it is still difficult to know how translation was established in the RNA world, it is likely that many of the steps in the process today are at least chemically similar to those that were initially occurring [START_REF] Ellington | Evolutionary origins and directed evolution of RNA[END_REF]). In the absence of aminoacyl tRNA synthetase, it can be conjectured that RNA self-aminoacylation constituted the early solution to the first chemical step of protein synthesis. This question is at the center of the present investigation. Ribozyme-catalyzed aminoacylation requires three basic elements: amino acids, energy resource and buffering conditions (van der Gulik and Speijer 2015). The availability of free amino acids may have been important already in the early history of life. Mixtures of some simple amino acids, such as glycine, alanine, aspartic and glutamic acid, are easily formed in classical Miller experiments [START_REF] Miller Sl | A production of amino acids under possible primitive earth conditions[END_REF]. These amino acids were likely among the first substrates of ribozyme-directed protein synthesis [START_REF] Orgel | Some consequences of the RNA world hypothesis[END_REF][START_REF] Da Silva | From the RNA world to the RNA/protein world: Contribution of some riboswitchbinding species?[END_REF]. Some of today's amino acids, such as lysine, arginine and tryptophan, are much more complex and were

plausibly not present at the origin.

An energy source is required to activate the amino acids and allow them to react with the 3'-end of an RNA. Under normal pressure and temperature conditions, this activation can be fulfilled with the participation of an energy-rich cofactor. Although ATP is the most common of such cofactors in present-day cells, there are some discussions about the presence of ATP in the prebiotic environment (van der Gulik and Speijer 2015), and there is no widely accepted theory about prebiotic activation. The presence of RNA molecules however implies the presence of activated nucleotides. Furthermore, RNA aptamer investigations demonstrated that strong interactions can occur between RNA and ATP molecules [START_REF] Morii | In vitro selection of ATP-binding receptors using a ribonucleopeptide complex[END_REF][START_REF] Vaish | A novel, modification-dependent ATP-binding aptamer selected from an RNA library incorporating a cationic functionality[END_REF][START_REF] Huang | Evolution of aptamers with a new specificity and new secondary structures from an ATP aptamer[END_REF][START_REF] Sazani | A small aptamer with strong and specific recognition of the triphosphate of ATP[END_REF]). Interestingly, most of in vivo RNA processing enzymes (RNA ligase, RNA spliceosome, aaRS, etc.) require ATP as cofactor to perform their functions. So far however, no in vitro selected ribozyme is capable of "utilizing"

ATP as an energy source for activation, and all know self-aminoacylating ribozymes require pre-activated amino acids as substrates. Yet pre-activated amino acids, such as aminoacyladenylate, are unstable molecules quickly hydrolyzed in solution [START_REF] Illangasekare | Small-molecule-Substrate Interactions with a Self-aminoacylating ribozyme[END_REF]. It is thus logical to speculate that early self-aminoacylating ribozymes could also manage the activation step. The quest for an activatingself-aminoacylating ribozyme constitutes a main challenge investigated in the present work.

Part I In vitro selection of ribozymes 1. Some aspects of in vitro selection and the SELEX methodology

In 1960s, Spiegelman and co-workers observed that the genomic sequence of the RNA bacteriophage Qβ can dramatically change during a repeated in vitro replications by the Qβ replicase [START_REF] Mills | An extracellular Darwinian experiment with a selfduplicating nucleic acid molecule[END_REF]. These changes are caused by the so-called "serial transfer amplification" during which the RNA of a late experiment is the result of previous RNA amplification reactions. After several rounds of replication, 90% of the RNA turns out to be truncated sequences. This directed evolution experiment is often cited to illustrate how Darwinian evolution could also occur at molecular level during in vitro experiments.

This experiment was the premise of the SELEX methodology first explored by Bartel and Szostak in the 90s [START_REF] Bartel | Isolation of new ribozymes from a large pool of random sequences[END_REF]. The main purpose of a SELEX procedure is to isolate oligonucleotide sequences (DNA or RNA) that have a special property being repeatedly selected from a large pool of random molecules during selection cycles. A successful design lead to a detectable enrichment of the pools with so-called active molecules, possibly after just a few cycles or up to 15-20 cycles.

In terms of ribozyme in vitro selection, the major established methodologies have been classified into three main categories [START_REF] Breaker | In Vitro Selection of Catalytic Polynucleotides[END_REF].

1) Distinction by self-modification. This method was used in many of the early in vitro selection experiments. Its goal is to isolate ribozymes based on self-modification. By means of physical separation (through molecular recognition), active variants can be selected depending on their size (like the ligating or cleaving ribozyme, Table 3) and/or modification (like the phosphorylation ribozyme, Table 4).

2) Binding to specific targets. This method has helped researchers to explore numerous RNA aptamers via their different affinities to the substrates, which includes proteins, amino acids, ATP and other chemical compounds. These aptamers can also work as probes to target specific substrates and closely-related compounds. They may play the role of inhibitors of metabolic processes or as diagnostic agents [START_REF] Joyce | In vitro evolution of nucleic acids[END_REF][START_REF] Ohkawa | Ribozymes: from mechanistic studies to applications in vivo[END_REF].

3) Altering catalytic properties. Instead of using pools of random molecules for the selection, some experiments explored the possibility to alter the catalytic function of existing ribozymes. By means of partial randomization or point mutations, researchers were able to engineer the efficiency of natural ribozymes and investigate their catalytic mechanism [START_REF] Kumar | Artificial evolution and natural ribozymes[END_REF].

An in vitro selection requires the specification of several parameters defining the SELEX protocol. One of the most important parameter is perhaps the length of the templates. The success of in vitro selection experiments critically depends on the design of the starting population of molecules; It requires the initial pools to provide enough molecular and structural diversity for the selection. In that respect, long random sequences can provide more structural possibilities. Long sequences however have a higher propensity to misfolded, and in any case full molecular diversity cannot be reached in test tubes with random track longer than about 28 bases [START_REF] Pobanz | Improving the odds: Influence of starting pools on in vitro selection outcomes[END_REF]. On the other hand, it has been demonstrated that (very) short random track implementing carefully design SELEX protocols may lead to a successful selection [START_REF] Turk | Multiple translational products from a five-nucleotide ribozyme[END_REF][START_REF] Pobanz | Improving the odds: Influence of starting pools on in vitro selection outcomes[END_REF]. There is thus no clear rule to establish this parameter. In their early successful attempt, Bartel and Szostak did use an extremely large initial RNA pool with 220 random nucleotides present on the template to select ribozymes with ligation activity [START_REF] Bartel | Isolation of new ribozymes from a large pool of random sequences[END_REF]. With a well-optimized SELEX protocol, Yarus and co-workers later found a ribozyme specified by as little as five nucleotide that is able to catalyze aminoacylation using only three nucleotides in the active center [START_REF] Turk | Multiple translational products from a five-nucleotide ribozyme[END_REF]. This result, as well as results obtained with self-aminoacylating ribozymes as small as about 25 nt [START_REF] Illangasekare | Specific, rapid synthesis of Phe-RNA by RNA[END_REF][START_REF] Lehmann | Efficiency of a self-aminoacylating ribozyme: effect of the length and base-composition of its 3' extension[END_REF]) constituted an argument for us to run a SELEX protocol using pools characterized by small random RNA of the order of 20 to 40

nucleotides.

An issue that is sometimes encountered during SELEX experiments can be mentioned here.

It can occur that so-called "selfish RNA" or "mini monsters" emerge and invade the sequence population of random pools subjected to selection. In most cases, selection starts with a pool of RNA with random regions flanked by constant regions that are required for PCR (Figure 7).

Due to their inherent abundance, some short nucleic acids originating from these constants tracks can invade the selection protocol and replicate more rapidly than others. These small RNA species may finally dominate the population of amplified molecules [START_REF] Breaker | Self-Production of Supramolecular Structures: From Synthetic Structures to Models of Minimal Living Systems[END_REF]. These false positive signals may overspread the targeted molecules and thus increase the difficulty to isolate active molecules. An in vitro SELEX experiment is the most powerful strategy so far established to uncover functional nucleic acids, either ribozymes (RNA) or DNAzyme (DNA). It is somehow like a natural selection achieved in a tiny controlled environment, under laboratory conditions. It can be mentioned that in silico selection, in which a molecular target can be tested by large numbers of error-and-trial binding tests with different molecules is also a rapidly growing field investigated by bioinformaticians [START_REF] Könnyű | In silico ribozyme evolution in a metabolically coupled RNA population[END_REF].

The selection of artificial ribozymes

The diversity of artificial ribozymes found by in vitro selection is highly variegated compare with natural ribozymes. In this section, we are going to categorize the artificial ribozymes based on their functions as well as the optimal condition(s) under which they are active. The conditions highlighted in yellow are considered in relation with our protocol to select active ribozymes capable of both amino acid activation (with ATP/GTP) and 3' transesterification.

Since self-amplification would be the beginning of RNA evolution, researchers were quite interested in studying RNA polymerization activity at the early stage of the SELEX development. When Sleeper and co-workers discovered that single nucleotides can aggregate and slowly polymerize [START_REF] Sleeper | The catalysis of nucleotide polymerization by compounds of divalent lead[END_REF], the question was: could RNA molecules rapidly increase in diversity and size, accumulate, and undergo some kind of natural selection?

In vitro selected ribozymes that can catalyze template-directed elongation are shown in Table 1. These ribozymes may represent the ancient semi-conservative replicators. The existence of several kinds of such aptamers suggests that RNA molecules could form binding pockets for amino acid activation with ATP and/or GTP. Known interactions between amino acids and RNA have been reviewed by Yarus and co-workers [START_REF] Yarus | RNA-amino acid binding: a stereochemical era for the genetic code[END_REF]. They may give us hints about the early aminoacylation of RNA and genetic code evolution.

Considering ribozyme families at large, self-processing ribozymes so far constitute the largest family of artificial ribozymes. This family comprises self-cleaving, phosphorylating and aminoacylating ribozymes.

Many in vivo selected self-cleaving ribozymes, such as the HDV ribozyme (Riccitelli and Luptá k 2013) and the hammerhead ribozyme [START_REF] Lee | Bridging the gap between theory and experiment to derive a detailed understanding of hammerhead ribozyme catalysis[END_REF] have been engineered by researchers to investigate and improve the efficiency of their catalytic mechanisms. Selfcleaving activity is based on the phosphate-ribose skeleton [START_REF] Jimenez | Chemistry and Biology of Self-Cleaving Ribozymes[END_REF]). An external electron donor attacks the hydrogen atom of a 2' hydroxyl group. The negatively charged 2' oxygen then becomes an active nucleophile that can attack the 3' phosphate and yield a transition intermediate (Figure 8). Sharing a similar acid-base catalytic mechanism, ribozymes catalyzing RNA ligation shows another interesting aspect of RNA catalysis (Table 3). This key principle of cleaving and ligating is supporting one of the basic catalytic functions of RNA molecule, and is a major reason why RNA molecule is less stable than DNA. RNA-catalyzed ligation and cleavage is the basis for RNA recombination and thus provide opportunities for evolution by increasing molecular diversity. [START_REF] Sassanfar | An RNA motif that binds ATP[END_REF][START_REF] Connell | Utilization of cofactors expands metabolism in a new RNA world[END_REF]. Aminoacylation by ribozymes has drawn a great attention from scientists during last two decades, and is at the center of our investigations. The aminoacyl RNA is the genetic information transporter and an essential tool for protein synthesis: this molecule is central for the evolution from the RNA world to the protein world. Scientists have already uncovered many ribozymes that could have an activity close to the original aminoacyl-RNA synthetase (Table 4). Besides, other functions, such as RNA alkylation, thiol ester formation and RNA capping, were also further discovered by in vitro experiments, revealing that RNA has a very large potential in catalytic activities [START_REF] Chen | Ribozyme catalysis of metabolism in the RNA world[END_REF]. Considering a broad image of catalysis in the RNA world (Figure 8), most of the ribozymes are active at a pH ranging from 6.5 to 8.5. Catalysis is facilitated by divalent cation.

This overview reveals another striking feature: it has not yet been possible to isolate ribozymes catalyzing amino acid activation at physiological pH. In this connection, previously in vitro isolated self-aminoacylating ribozymes all require highly concentrated AMP-activated amino acid at the start of the experiments to compensate for its rapid decay at physiological pH (the half-life of Phe-AMP at pH 7 is only a few minutes at 0º C). How is this issue managed in modern organisms? tRNA aminoacylation in vivo is achieved by a series of enzymes called aminoacyl-tRNA synthetase (aaRS). The first step of aminoacylation is amino acid activation with ATP, yielding the activated amino acid (aa-AMP), which remains bound to the enzyme.

The aaRS subsequently catalyzes the transfer of the amino acid to the 3'-end of the tRNA (transesterification). In early in vitro SELEX experiments, researchers successfully isolated 2' or 3' self-aminoacylating ribozymes using synthetic aa-AMP (or with other activating cofactors) as substrate. The question is: where did those activated amino acids come from in the early RNA world environment? Given that transesterification best works at physiological pH, and because aa-AMP is highly unstable at that pH, it can be speculated that, similarly to the aaRS, ribozymes that could successfully manage the activation step should also be capable of catalyzing transesterification. In other words, an aminoacylating ribozyme that could sustainably achieve aminoacylation must be able to manage both chemical steps at the same pH.

Uncovering ribozymes that could work as primary aaRS is our main goal. It would fill a huge gap of RNA world hypothesis.

Part II Identification of aminoacylated RNA from total RNA

Transfer RNA in vivo

As discussed in last section, RNA-catalyzed RNA aminoacylation is the key chemical reaction in the evolution of life from the late RNA world to the modern DNA/protein world. In modern cellular life, tRNA as the major aminoacylated RNA is well known as the key actor of mRNA translation. Besides, tRNA also perform additional functions in gene regulation [START_REF] Raina | TRNAs as regulators of biological processes[END_REF].

The control and regulation of tRNA aminoacylation is one of the well studied global regulatory system in bacteria. At the translational level, enzymes like RelA in E. coli can sense the stalling of protein synthesis that occurs when uncharged tRNA start accumulating due to amino acid starvation. These enzymes activate a downstream gene regulation network to inhibit global gene transcription and activate some other genes related to amino acids synthesis (Figure 9, Left) [START_REF] Ross | The Magic Spot: A ppGpp Binding Site on E. coli RNA Polymerase Responsible for Regulation of Transcription Initiation[END_REF]. Another aminoacylated RNA playing an important role at the translational level is the transfer-messenger RNA (tmRNA), a bacterial RNA with tRNA-like and messenger RNA-like motifs [START_REF] Komine | A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli[END_REF]. The main function of tmRNA is to rescue the translation system by: 1) rescuing the stalled ribosome, 2) degrading the problematic mRNA, and 3) extending the unfinished polypeptide [START_REF] Janssen | The tmRNA ribosome-rescue system[END_REF]. This translation quality control system is involved in bacterial development, pathogenesis and stress responses. At the transcriptional level, the level of tRNA aminoacylation can regulate the expression of genes involved in amino acid metabolism through the T-box regulatory system in Gram-positive bacteria [START_REF] Green | The T box mechanism: tRNA as a regulatory molecule[END_REF]. A 5' leader T-box riboswitch is able to bind both the anticodon and the NCCA 3'-end of an uncharged tRNA when this population is high. This results in a readthrough of the termination site and the transcription of the full length mRNA (Figure 9, Right) (Henkin 2008). Most of the genes regulated by tRNA are directly involved in translation, and crucial for survival (Figure 10). An external stress can cause changes in tRNA gene expression, aminoacylation status, even base modification of specific tRNAs, which in turn regulate other genes at both transcriptional and translational levels [START_REF] Ibba | Transfer RNA comes of age[END_REF]. It is thus desirable to understand how changes like aminoacylation level and base modifications affect gene regulation. Researchers have developed some deep sequencing protocols [START_REF] Guo | Transfer RNA detection by small RNA deep sequencing and disease association with myelodysplastic syndromes[END_REF][START_REF] Diebel | Beyond the Ribosome: Extra-translational Functions of tRNA Fragments[END_REF] to analyze tRNA expression profiles from total RNA. A major difficulty that still prevents a quantitative and qualitative overview of these profiles is the presence of modified bases on RNA, and tRNA molecules are heavily modified post-transcriptionally [START_REF] Balke | Landmarks in the Evolution of (t)-RNAs from the Origin of Life up to Their Present Role in Human Cognition[END_REF]. More than 100 kinds of base modifications have been found so far according to the RNA modification database (http://mods.rna.albany.edu/mods/). These base modifications can increase the molecular stability and regulate the affinity of codon-anticodon interaction [START_REF] El Yacoubi | Biosynthesis and function of posttranscriptional modifications of transfer RNAs[END_REF]. Because they can cause stops/pauses of the reverse transcription enzyme, it is not always possible to obtain full length cDNA from these RNA, and that generates biases in RNAseq analysis. Interruption events due to modifications have already been investigated [START_REF] Motorin | Identification of modified residues in RNAs by reverse transcription-based methods[END_REF]). Furthermore, mass spectrometry methods are now available to detect base modifications [START_REF] Basturea | Research Methods for Detection and Quantitation of RNA Modifications[END_REF].

Deep sequencing analysis of total RNA have revealed new features related to tRNA degradation: a recently discovered new class of small RNA are the so-called tRNA-derived small RNA fragments (tRFs). These small fragments result from the processing of mature or precursor of tRNAs by RNase P, RNase Z and Dicers , that generates tRNA halves, 5' tRF, 3' CCA tRF, 3' U tRF and 5' leader exon tRFs structures (Figure 11) [START_REF] Lee | A novel class of small RNAs : tRNA-derived RNA fragments ( tRFs ) A novel class of small RNAs : tRNA-derived RNA fragments[END_REF].

Figure 11. tRNA-derived small RNAs (tRFs). Precursor tRNAs need to be processed by RNase P, RNase Z at both 5'and 3'-ends, as well as CCA-adding enzyme to yield the mature tRNA in the nucleus. Processed fragments from pre-tRNA and mature tRNA is possible to play a role as small RNA in cell. tRNA can further degrades into tRNA halves, 5' tRF, 3' CCA tRF, 3' U tRF and 5' leader exon tRFs, etc. Dash lines and question marks emphasize the unknown mechanisms of formation or transport of these tRFs [START_REF] Raina | TRNAs as regulators of biological processes[END_REF].

tRFs discovered by high throughput sequencing constitute a class of short RNAs that are second most abundant to microRNAs. These small RNA can be categorized by their size and processing sites. They seem to be involved in many gene regulation and are related to some diseases [START_REF] Garcia-Silva | Hints of tRNA-derived small RNAs role in RNA silencing mechanisms[END_REF][START_REF] Fu | Small Non-coding Transfer RNA-Derived RNA Fragments (tRFs): Their Biogenesis, Function and Implication in Human Diseases[END_REF]. Several facts are already know about tRFs [START_REF] Kanai | Disrupted tRNA Genes and tRNA Fragments: A Perspective on tRNA Gene Evolution[END_REF]: 1) They do not derive from abundant cellular tRNAs, and the numbers of tRFs do not correlate with the parental tRNA gene copy numbers; 2) their fragmentation patterns are depended on their anticodons; 3) the fragmentation patterns can be changed based on environmental conditions; and 4) some tRFs are bound to Argonaute/Piwi proteins (RNAinduced silencing complex). tRFs have been observed in all three domains of life and are expressed by different pathways [START_REF] Keam | tRNA-Derived Fragments (tRFs): Emerging New Roles for an Ancient RNA in the Regulation of Gene Expression[END_REF].

Other possible aminoacylated RNA?

The above overview shows that new features related to tRNA metabolism are still being discovered. Our interest in RNA aminoacylation led us to ask the following question: besides tRNA and tmRNA, are there other types of RNAs that are aminoacylated for functional purposes in modern cells? Some clues suggest this possibility. Shi and co-workers have investigated several truncated tRNA tetraloops that can be recognized and aminoacylated by aaRS in vitro [START_REF] Shi | RNA tetraloops as minimalist substrates for aminoacylation[END_REF]). These tetraloops are derived from tRNA acceptor arm (Figure 12), and one may wonder whether some still unidentified tRFs or other RNA could undergo aminoacylation.

Figure 12. tRNA derived tetraloop with minihelix structure that can be recognized and aminoacylated by aaRS.

Very interestingly, some tRNA-like small RNAs have been identified in eukaryotic cells.

For example, human and rat U2snRNAs are small RNAs that involved in mRNA splicing, which contain a 3'-end that strongly resembles a tRNA minihelix (the 'top half ' of tRNA) including a CCA ending [START_REF] Geslain | Regulation of RNA function by aminoacylation and editing?[END_REF]. Chen and co-workers reported a widespread bacterial noncoding RNA, Y RNAs, that mimics tRNA T-loop and D-loop structures and bear some base modifications similar to that of tRNA. In Mycobacterium smegmatis, Y RNA can be processed by RNase P and further modified by CCA adding enzyme [START_REF] Chen | Bacterial noncoding Y RNAs are widespread and mimic tRNAs[END_REF]. They suggest that this kind of RNA may mimic tRNA behavior to avoid rapid degradation by RNase in vivo. If so, is it possible that this tRNA-like Y RNA could also be aminoacylated? Furthermore, some RNAs found at the 3'-end of plant viruses genome mimic host tRNA structures to support infectivity [START_REF] Fechter | Novel features in the tRNA-like world of plant 129 viral RNAs[END_REF][START_REF] Hema | Repair of the tRNA-Like CCA Sequence in a Multipartite Positive-Strand RNA Virus[END_REF][START_REF] Dreher | Role of tRNA-like structures in controlling plant virus replication[END_REF]).

All these facts suggest that RNA in vivo aminoacylation might be more widespread than previously thought. They motivated us to develop an RNAseq protocol specifically designed for the identification of 3' aminoacylated RNA from total RNA. Establishing such a protocol could not only reveal new types of aminoacylated RNA; it will also provide additional information on tRNA regulation and its connection with cell metabolism.

Chapter I Development of a SELEX method to uncover autoaminoacylating ribozymes

Introduction

The SELEX methodology, introduced in the first section, is a technique that allows the screening of pools of random RNA or DNA for molecules with a particular feature. In order to introduce and best justify our protocol of selection of aminoacylated RNA, it is appropriate to present some successful in vitro selection experiments that already allowed the isolation of SELEX experiments have allowed at least three research groups (Szostak, Yarus and Suga)

to successfully isolate ribozymes involved in the aminoacylation reaction. The two reactions involved in the RNA aminoacylation from free amino acids are the following:

1) Amino acid (aa) activation: aa + ATP  aa-AMP + ppi

2) 3' end transesterification: aa-AMP + RNA  aa-RNA + AMP These two reactions are coupled by the aaRS in the modern translation system, a thermodynamical requirement due to the very low equilibrium constant of the first reaction.

Activation is driven forward by the high affinity of the aaRS for aa-AMP and by the coupled second reaction. Contrary to activation, transesterification has indeed a very high equilibrium constant: the free-energy of aa-AMP hydrolysis is about -15 kcal/mol at pH 7.0 and 20º C [START_REF] Pascal | From the Prebiotic Synthesis of α-Amino Acids Towards a Primitive Translation Apparatus for the Synthesis of Peptides[END_REF], resulting in an overall equilibrium constant (reactions 1 and 2) slightly lower than 1 (Berg et al.1961). These thermodynamic considerations show that the difficulty essentially lies in the completion of the activation reaction. Not surprisingly, almost all successful SELEX experiments so far published concern transesterifying ribozymes using pre-activated amino acids as substrates. It is quite illuminating to examine the structure of these identified ribozymes (and related ones) because they provide indications about critical requirements for aminoacylation, and thus for the design of the random pools to implement the SELEX protocol.

Design of the random pools

An overview of the published results reveals that the templates designed by different investigators are similar. In general, it includes 50 or 80 random bases flanked by around 20 conserved nucleotides for PCR amplification, which provides around 10 14 ~10 15 different sequences in the initial pools (under an appropriate concentration of ~1 M). In these former selections, the activity of the isolated ribozymes had to be compatible with these constant tracks, but this issue is often not much discussed. However, these ribozymes have particular 5' and 3' ends that are usually most critical for the activity. Almost all of them have structured bases at the 5'-end and a more flexible, single-stranded 3'-end (Figure 1-2) [START_REF] Illangasekare | Aminoacyl-RNA synthesis catalyzed by an RNA[END_REF][START_REF] Lohse | Ribozyme-catalysed amino-acid transfer reactions[END_REF][START_REF] Lee | Ribozyme-catalyzed tRNA aminoacylation[END_REF]). An initial 95-nt "#29" ribozyme isolated by [START_REF] Illangasekare | Small-molecule-Substrate Interactions with a Self-aminoacylating ribozyme[END_REF] (Figure 12C) could be shrinked to a functional 29-nt ribozyme (Figure 123A), revealing that the essential part involved in catalysis is quite small.

Furthermore, a study of the 3'-end base-composition of this "29 nt" ribozyme shows that the aminoacylation efficiency can be tuned by changing just a few nucleotides [START_REF] Lehmann | Efficiency of a self-aminoacylating ribozyme: effect of the length and base-composition of its 3' extension[END_REF]). On the 5' side, it was shown that a terminal triphosphate (always present with T7 transcripts) is essential to the activity of this ribozyme family (Figure 123A). Altogether, these observations suggest that it is crucial to minimize the (arbitrary) constant tracks at both 3' end 5' ends in the design of the random pools. A, B and C are adopted from [START_REF] Illangasekare | Aminoacyl-RNA synthesis catalyzed by an RNA[END_REF][START_REF] Lohse | Ribozyme-catalysed amino-acid transfer reactions[END_REF][START_REF] Lee | Ribozyme-catalyzed tRNA aminoacylation[END_REF].

The above conclusions most likely triggered Yarus and co-worker to try an improved SELEX protocol, with which they could identify much smaller self-aminoacylating ribozymes from initial pools characterized by entirely random 3' ends. Working with this protocol, they found a series of ribozymes that catalyze aminoacylation with only three conserved nucleotides (Figure 1-3, B) [START_REF] Chumachenko | Rapid and Simple Ribozymic Aminoacylation Using Three Conserved Nucleotides[END_REF]). The identified ribozymes had an 8-base junction domain (Figure 1-3, B, Red rectangle) present in the middle of the sequence. These candidates could be simplified into a 9-nt "GUGGC/GCCU" base paired ribozyme that can achieve aminoacyl-, peptidyl-, dipeptidyl-reactions when highly concentrated Phe-AMP is present [START_REF] Turk | Catalyzed and spontaneous reactions on ribozyme ribose[END_REF]. Similar to the previously selected ribozymes, the 5'-end is structured. The truncated C3 ribozyme (Figure 123C) however has an unstructured 5'-end.

(Figure 1-3, C)
The structural stability of the active site is achieved by setting a low temperature (4º C) during incubation.

These former investigations provided unevaluable information that guided us in the design of our random pools. Prominent considerations are the following:

1) Although full randomization is desirable, the 5'-end sequence of the pools may not require to be strictly randomized. T7 transcript must all start with 5' GG for an efficient transcription, a constraint that seems to be an advantage in terms of secondary structure stability.

2) The 3'-end sequence of the pool is critical for transesterification: maximizing randomization may allow our SELEX experiment to have access to a higher diversity of ribozymes. This is especially important since we seek to isolate ribozymes with dual catalytic activity (activation and transesterification).

3) The size of the random track may not need to be extremely large. Small motifs can be sufficient for catalysis (Figure 1-3).

Incubation conditions and activating cofactor(s)

The choice of particular incubation conditions set to promote ribozyme aminoacylation may be guided by the examination of the chemistry being involved, and by previous studies on such ribozymes.

Transesterification is based on a classic "acid-base" chemistry mechanism [START_REF] Hatano | Lanthanum(III) catalysts for highly efficient and chemoselective transesterification[END_REF]. A Lewis acid coordinates with the oxygen of a carboxyl group to reduce the electron density of the carbon atom. The 2' or 3' oxygen atom of an RNA attacks the carbonyl group, and the leaving group is subsequently released (Figure 1 At least three aspects have to be considered for aminoacylation: 1) the presence of a Lewis acid, 2) the nature of the activating cofactor and 3) setting an optimal pH.

Lewis acid usually refers to cations like Na + , K + , Mg 2+ and Ca 2+ . Are these cations always required for aminoacylation activity? In an early study on a 5'-self capping ribozyme, the Yarus group discovered that a selected "isolate 6 RNA" is a Calcium dependent ribozyme while the presence of Magnesium even inhibits catalysis [START_REF] Huang | 5'-RNA self-capping from guanosine diphosphate[END_REF]. On the other hand, the later investigated tiny ribozyme "GUGGC/GCCU" (Figure 1-3, C) does not require divalent cations at all; the presence of Magnesium only slightly increases the reaction rate [START_REF] Turk | Catalyzed and spontaneous reactions on ribozyme ribose[END_REF]). Nevertheless, some ribozymes such as the Yarus "#29 isolate" (Figure 1-3, B) requires both Calcium and Magnesium for activity, while other isolates show different divalentdependence profiles [START_REF] Illangasekare | Aminoacyl-RNA synthesis catalyzed by an RNA[END_REF]. In addition of being potential Lewis acids, divalent cation helps RNA molecule to properly fold by neutralizing the negative charge of polyanionic backbone and support out-sphere binding to stabilize the secondary structure [START_REF] Saito | Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme[END_REF][START_REF] Derose | Metal ion binding to catalytic RNA molecules[END_REF][START_REF] Sigel | Alternative roles for metal ions in enzyme catalysis and the implications for ribozyme chemistry[END_REF]. From these results, a conservative strategy is to include both Magnesium and Calcium in the incubation buffers in addition of monovalent cations.

Because a carboxyl group of an amino acid may not react with a hydroxyl group under standard conditions due to a very high energetic barrier [START_REF] Zhang | Peptide bond formation by in vitro selected ribozymes[END_REF][START_REF] Li | Ribozyme-catalyzed aminoacylation from CoA thioesters[END_REF][START_REF] Murakami | Aminoacyl-tRNA synthesis by a resin-immobilized ribozyme[END_REF][START_REF] Niwa | A flexizyme that selectively charges amino acids activated by a water-friendly leaving group[END_REF][START_REF] Morimoto | Flexizymes: their evolutionary history and the origin of catalytic function[END_REF], former studies on

A B G14 U15 U26
aminoacylating ribozymes all used pre-activated amino acids to allow transesterification. The commonly employed leaving group, such as AMP, CoA (Coenzyme A), CME (Cyanomethyl ester), CBT (p-chlorobenzyl thioester), DBE (3,5-dinitrobenzyl ester), etc., are all produced by organic synthesis. All these activated forms may easily react, but some of them (such as AMPaa) have very short lifetimes and are quickly hydrolyzed in solution. In terms of efficiency, a system implemented with ribozymes requiring pre-activated amino acids as substrates would be extremely inefficient and unlikely to occur in a primitive environment. It is thus reasonable to conceive the possibility that some still unknown ribozymes could manage both activation and aminoacylation.

Choosing an appropriate pH is another critical issue. The aminoacylation reaction requires physiological pH for optimal activity; a higher pH will reduce the half-life of active acyl bond (like Phe-AMP) and also increase the instability of the aminoacyl-RNA ester bond. A low pH will reduce nucleophilic activity of 2'-/3'-hydroxyl groups [START_REF] Murray | A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction[END_REF], a phenomenon clearly established in the case of ribozymes isolated by Yarus and coworkers [START_REF] Illangasekare | Small-molecule-Substrate Interactions with a Self-aminoacylating ribozyme[END_REF].

Is low pH a requirement for amino acid activation?

The ribozyme-catalyzed activation of amino acids remained an unsolved problem for many years since the discovery of the first aminoacylating ribozymes in 1995. In 2001, Yarus and coworkers came up with another breakthrough in the ribozyme field with the identification of an RNA (called KK13; Figure 12345A) able to catalyze amino acid activiation by using the triphosphate group at the 5'-end of the RNA. The pH was set to 4.0 during selection, and the isolated ribozyme, that requires Calcium, was also found to best work at that pH [START_REF] Kumar | RNA-catalyzed amino acid activation[END_REF]. This is so far the only known RNA that can catalyze carboxyl group activation.

A B

Because this ribozyme functions at pH 4.0 while aminoacylating ribozymes are most efficient at pH 7.0, a combination of both reactions could not be envisaged in a same aqueous media. After an unsuccessful attempt to isolate RNA with both catalytic activities at the same pH, Xu and co-workers established a smart experimental design to combine two ribozymes and two reaction conditions to successfully achieve amino acid activation and aminoacylation [START_REF] Xu | RNA aminoacylation mediated by sequential action of two ribozymes and a nonactivated amino acid[END_REF]. In this strategy, an amino acid is first activated with the KK13 ribozyme. They then re-designed the C3 ribozyme to allow base pairing with the 5' end of the KK13 ribozyme, that yielded a truncated C3a (Figure 12345B). The C3a variant enables the activated amino acid to be close to the U26:U15G14 catalysis core of C3a, and thus promote transesterification. This work is meaningful because this is the first time that researchers successfully produce aminoacylated RNA by only using RNA as catalytic cofactor.

However, these experiments can only occur sequentially. The KK13 ribozyme is only functional at pH=4 while transesterification happens at physiological pH range. The reason for which a pH of 4.0 was chosen in the selection that yielded KK13 is that acetate groups cannot react with a triphosphate at physiological pH if a positive charge is not around for coordination A fundamental problem stands here. If we provide enough positive charges during in vitro selection, is it possible to achieve amino acid activation at physiological pH and thus work in conditions that are optimal for aminoacylation? Seeking for a ribozyme that can truly functions as a primary aaRS is the significant challenge investigated in our work.

Experimental design

Overview of the protocol

The protocol is divided into two parts. The first part consists of the incubation of the random RNA pool in a solution comprising a mixture of 19 amino acids, activating cofactors (ATP and GTP) and various monovalent and divalent cations. This incubation is designed to promote selfaminoacylation on active RNA molecules of the pool. In a second part, a procedure of oxidation and deacylation followed by adaptor ligation at the 3'-end and RT-PCR is performed to selectively amplify aminoacylated RNAs. 1. The SELEX experiment starts with the synthesis of a pool of RNA in vitro transcribed from a series of cDNA templates with 10 to 30 random positions flanked by very short constant tracks.

2. After incubation in a buffer with the amino acids, ATP/GTP, different ions and polymers, the mixture of RNA is collected for selection.

3. The selection step has two parts: First, RNA is incubated in a solution of sodium periodate to oxidize the 3'-end of molecules with no aminoacyl ester attached to them. Periodate oxidation as a popular strategy has been used in molecular biology to oxidize the cis-diols groups at the 3'-end of RNA. It is also applied in many RNA in vitro experiments to distinguish the aminoacyl and non-aminoacyl tRNA, introduce external modified bases into RNA, or to detect post-transcriptional modifications [START_REF] Zaborske | Genomewide analysis of tRNA charging and activation of the eIF2 kinase Gcn2p[END_REF][START_REF] Murakami | Aminoacyl-tRNA synthesis by a resin-immobilized ribozyme[END_REF][START_REF] Kurata | Quick two-step RNA ligation employing periodate oxidation[END_REF][START_REF] Kawano | Novel small noncoding RNAs in mouse spermatozoa, zygotes and early embryos[END_REF]. Any RNA with free 3'-cis-diol group of the ribose is oxidized into di-aldehyde groups, while an RNA aminoacylated at the 3'-end is protected from oxidation. Subsequently, deacylation releases the amino acids from these RNA, making the diols at the 3'-end available to adaptor ligation during the next step.

4. RNA with free non-oxidized 3'-end ligate with a 5'-phosphorylated RNA adapter carrying a specific restriction site near the 5'-end junction. (Adapters design see Section 2.5)

5. Reverse transcription generates a cDNA that can readily undergo another ligation step.

6. A double-stranded DNA anchor carrying the T7 promoter sequence ligates with the cDNA using a few overhanged bases for base paring. (This step is further optimized in the final protocol. See details in Section 2.6)

7. The fully structured cDNA including a T7 promoter and an adapter sequence with selected target in between is ready for PCR amplification.

8. The treatment with a restriction enzyme removes the adapter part. A new pool of RNA is transcribed from these templates and allows to start a next selection cycle.

Template design

In the process of establishing a SELEX procedure to isolate active RNA molecules, the length of the random track and that of the conserved regions of the RNA templates constitute a critical issue.

The size of the random track in most in vitro SELEX experiments varies from 20 to 200 nucleotides [START_REF] Ruckman | 2′-Fluoropyrimidine RNA-based Aptamers to the 165-Amino Acid Form of Vascular Endothelial Growth Factor (VEGF165): INHIBITION OF RECEPTOR BINDING AND VEGF-INDUCED VASCULAR PERMEABILITY THROUGH INTERACTIONS REQUIRING THE EXON 7-ENCODED DOMAIN[END_REF][START_REF] Lee | Ribozyme-catalyzed tRNA aminoacylation[END_REF][START_REF] Kumar | RNA-catalyzed amino acid activation[END_REF][START_REF] Thiel | Nucleotide bias observed with a short SELEX RNA aptamer library[END_REF].

In theory, RNA pools with more random positions provide a higher diversity and more possibilities to the selection, but this strategy results in less copies of each unique sequence present among the whole population at a given molar concentration [START_REF] Yarus | Chemical biology: Bring them back alive[END_REF]. The issue of how the length of initial pool influent the in vitro selection has been recently reviewed by

Pobanz and co-workers (Pobanz and Lupták 2016). Another aspect to integrate is the efficiency of the T7 RNA polymerase. This enzyme is more efficient in the transcription of long sequences compared to shorter ones, the lower limit being around 20 nucleotides. Long transcripts provide more structurally diverse binding sites but they may more easily miss-fold during the renaturation step in each selection cycle. Short transcripts can possibly provide active molecules, but set limits to the possible structures of an active site. It has been suggested that autocatalytic RNA networks are more likely to begin from monomers accumulation and short substrate interaction [START_REF] Gilbert | The RNA world[END_REF][START_REF] Yarus | Chemical biology: Bring them back alive[END_REF][START_REF] Higgs | The RNA World: molecular cooperation at the origins of life[END_REF]. Considering all these aspects, we decided to implement a selection protocol with 4 pools of RNA molecules with a size of 19, 23, 30 and 37 nucleotides while maximizing the relative portion of the random tracks.

Initial design

Our initial design was rather simple. In order to minimize the burden of the constant tracks at both 5' and 3' ends of the molecules, we choose to work with almost completely random RNA sequences, except for two Gs at the 5'-end, required for T7 transcription (Table 2-1). Studies on a 3'-end self-aminoacylating ribozyme have indeed shown that the length and the base composition of the 3'-end extension have a significant effect on their activities [START_REF] Illangasekare | Aminoacyl-RNA synthesis catalyzed by an RNA[END_REF][START_REF] Lehmann | Efficiency of a self-aminoacylating ribozyme: effect of the length and base-composition of its 3' extension[END_REF]). Furthermore, the composition of the 5'-end of these RNAs, including the presence of a 5'-triphosphate, may also dramatically impair the active site of this type of ribozyme [START_REF] Illangasekare | Small-molecule-Substrate Interactions with a Self-aminoacylating ribozyme[END_REF].

One major difficulty in the protocol related to the randomness of the RNA sequences is the specificity of reverse transcription. Random region can cause miss priming during the annealing step of reverse transcription. The final PCR could easily yield primer dimers as by-products.

We had to test different RNA adapters to solve this issue (Section 2.5).

Another main problem associated with these random libraries is the difficulty to keep the size of the pools constant. Because the T7 RNA polymerase has non-template extension activity and may drop off the template before the end of transcription, the size of the RNA pool will turn highly heterogeneous already after one or two cycles [START_REF] Chumachenko | Rapid and Simple Ribozymic Aminoacylation Using Three Conserved Nucleotides[END_REF]). In the worst cases, up to 50% of the in vitro transcribed RNA may contain so-called n+1 and n+2 nt sequences [START_REF] Milligan | Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates[END_REF][START_REF] Krupp | RNA synthesis: strategies for the use of bacteriophage RNA polymerases[END_REF]). T7 RNA polymerase can also bypass up to ~20 nucleotides gaps before resuming transcription [START_REF] Zhou | T7 RNA polymerase bypass of large gaps on the template strand reveals a critical role of the nontemplate strand in elongation[END_REF]. These events are clearly visible after selection during gel analysis, where smears are observed instead of clear bands. Another major difficulty related with the amplification of essentially random RNA sequences is the requirement of an adaptor ligation at the 5'-end of the transcripts. In order to prevent side-products of RNA ligation from occurring, this second ligation was attempted on cDNA after reverse transcription. Only two C residues could specify the 3'-end of these cDNAs.

We attempted to ligate them with a DNA anchor (Table 2-3, orange symbol) containing a 3'end overhang of two Gs constituting sticky end. Despite all our attempts in various conditions, we could not succeed in obtaining a final construct that could undergo a final PCR. It turns out that the yield of this final ligation step was always too low, and that other ligation events would always prevent us from obtaining the desired PCR product. Especially, the DNA anchor would ligate to the excess of primer of the adapter (Table 2-3) and generate primer-dimer as the majority PCR product. Probably, only two G residues 3' overhang could easily cause unspecific ligation between the DNA anchor and the primer of the adapter even if they were designed not to be compatible with each other. After months of unsuccessful attempts, we decided to abandon the idea of RNA transcripts with almost totally random 5'-end.

We re-designed the 5'-end while designing more constant positions in order to promote specific DNA ligation. We assumed that a DNA anchor mainly constituted by purine residues would conflict with the purine region of the primer of the adapter and prevent unspecific DNA ligation from happening between these two species. A control during which the whole procedure was tested without template (i.e. only with the primer of the adaptor and the DNA anchor) generated a strong by-product on the gel (Figure 2-2) with a length corresponding to a primer-dimer. After several unsuccessful attempts, we finally abandoned the strategy of applying two ligations (see Section 2.6).

Figure 2-2. By-products test: the left lane shows the primer dimer yield by a control which had processed the whole procedures with only the primer of the adapter and DNA anchor; the right lane is a PCR negative control with only two PCR primers.

Final design

In order to successfully achieve the final PCR amplification, we finally opted for a solution with a small stretch of constant positions at the 5'-end of the sequences of the pools. This constant track would allow the overhang of a forward primer to prime directly on the cDNA without the need of a second ligation. Although the arbitrary choice of these constant positions could be critical to the success of our SELEX protocol, we could not conceive any other feasible option.

We tested other methods to add the T7 adapter to the 5'-end of RNA template by modifying the overhang of DNA anchor, one step cDNA elongation and poly-A tailing, and they all failed. A new survey of the literature made us later realize that other groups were likely already confronted with this issue. In a recent SELEX investigation whose aim was to isolate new selfaminoacylating ribozymes, Yarus and coworkers also opted for a constant track at the 5'-end, while the 3'-end was completely random [START_REF] Chumachenko | Rapid and Simple Ribozymic Aminoacylation Using Three Conserved Nucleotides[END_REF]). They however did not try to minimize the size of the 5' track, and kept as much as 20 constant positions. Our carefully re-designed 5'-end contains 7 conserved bases (Table 2-4) that turned out to be long enough to ensure a correct priming during PCR with the overhang of a T7 primer. This "Short primer PCR" method to avoid primer dimer aggregation is introduced in detail in Section 2.7. Shorter constant tracks were tested, and could not allow the PCR amplification of our construct.

A final refinement in the design of our pools concerned the choice of the terminal 3' residues. Our initial 3'-end design was "UCR", where R (purine) stands for A or G (Table 234).

The rationale for a purine at the 3'-end is that they may bind RNA more strongly than a pyrimidine, and thus could better stabilize an active conformation of the 3'-end nearby a catalytic site. Also, the 3'-ends of modern tRNA bear a terminal adenosine, which may be an indication of such requirement. By using different overhang primers of the adapter during our test experiments, we observed a much stronger amplification of "UCA" compared with "UCG" in the final PCR, possibly because the "UCA" ending is more compatible at the RNA ligation level (Figure 2-3).

For this reason, the UCA ending was selected. The final design of our pools is shown in Table 2-5. To establish the total length of the initial pools of RNA, more than 50 different cDNA templates were preliminarily tested (data not show). Since the T7 polymerase has a rather low yield with DNA templates shorter than 20 nt, we chose 19 nt as the minimum size for one of these pools. Meanwhile, our "Short primer PCR" strategy is not suitable for amplifying cDNAs longer than 70 nt. Furthermore, it is reasonable to keep the length below 50 nt. When we prepare the initial pool of random RNA, the optimal amount of initial cDNA template we used for T7 transcription (0.1 nmol) includes 6.02×10 13 sequences, corresponding to the total diversity obtained with 23 random positions (4 23 = 7×10 13 ).

Composition of the incubation buffers for aminoacylation

Cofactors, such as ions, amino acids, peptides, ATP/GTP, nucleic acids, even water, are highly essential for ribozyme's structural folding and chemical catalysis. Natural ribozymes, such as Group II intron, RNase P and HDV ribozyme, all require an appropriate pH value as well as a certain amount of Mg 2+ to be active. Group I intron additionally requires GTP as cofactor in the first step of the splicing reaction. In our experimental design, several cofactors were added as components of the cocktail buffer to stimulate aminoacylation catalysis.

Since the "Primordial soup" theory was firstly proposed in 1924, the original life environment has been investigated in laboratories (Bailey 1938b). The negatively charged RNA chain requires the presence of various cations for proper folding and catalytic activity. In cooperation with different functional groups such as carbonyl, amino, hydroxyl, sulfanyl and phosphate, they may assist align the nucleophile with the reactants by reducing entropic barriers [START_REF] Sigel | Alternative roles for metal ions in enzyme catalysis and the implications for ribozyme chemistry[END_REF][START_REF] Schnabl | Controlling ribozyme activity by metal ions[END_REF].

Ions

Monovalent cations such as Li + , Na + , K + and NH4 + , are the most abundant ions in both the organisms and the natural environment. Monovalent cations are not only required for the ribozymes to adopt a proper folding [START_REF] Woodson | Metal ions and RNA folding: A highly charged topic with a dynamic future[END_REF][START_REF] Jiang | Monovalent cations use multiple mechanisms to resolve ribozyme misfolding[END_REF]) but also possibly responsible for their catalytic functions [START_REF] Murray | The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone[END_REF][START_REF] Hanna | Metal ions in ribozyme folding and catalysis[END_REF]. A recent research investigating crystal structures of the hammerhead ribozyme derived from Schistosoma mansoni has demonstrated that monovalent cations such as Na + could directly and specifically replace a divalent cation in the active site [START_REF] Anderson | Active-Site Monovalent Cations Revealed in a 1.55-Å-Resolution Hammerhead Ribozyme Structure[END_REF]). These coordinated monovalent ions could contribute to the ribozyme catalysis as a Lewis acid to promote deprotonation of OH group or to neutralize the negative charge on the phosphoryl oxygen of the transition state [START_REF] Ke | Structural Roles of Monovalent Cations in the HDV Ribozyme[END_REF]). Characterized by low atomic radius and charge density, monovalent cations are however far less efficient than the divalent ones; the HDV ribozyme requires up to 2-5 M of monovalent salts instead of Mg 2+ to support efficient cleavage [START_REF] Perrotta | HDV ribozyme activity in monovalent cations[END_REF].

Obviously, such a high concentration is hardly achieved in natural environment. Thus certain divalent cations have to be introduced into the selection to ensure the activity of the ribozymes.

The monovalent composition of our incubation buffer is 100 mM Na + and 100 mM K + , which is close to the concentration range of usual enzymatic reactions and organism cellular living environment [START_REF] Linzell | Intracellular concentrations of sodium, potassium and chloride in the lactating mammary gland and their relation to the secretory mechanism[END_REF]. Divalent cations, mostly Mg 2+ , Ca 2+ , Zn 2+ , Fe 2+ and Cu 2+ , together with RNA/DNA may have played an important role in the catalytic reactions relevant to the synthesis of prebiotic macromolecules during the early evolution of life. Even in the modern biology world, one in third of enzymes known so far are metalloenzymes [START_REF] Holm | Structural and Functional Aspects of Metal Sites in Biology[END_REF]. Magnesium, the second enriched divalent cation in the biosphere, almost dominates the biological system. Among the common divalent cations, Mg 2+ has small ionic radius (Mg 2+ = 0.72Å, Ca 2+ = 0.99Å, Na + = 0.95Å, K + = 1.52Å), a high charge density and high solubility at neutral pH [START_REF] Bowman | Cations in charge: Magnesium ions in RNA folding and catalysis[END_REF]. It can efficiently neutralize the negative charge of the electron donors, like oxygen, Calcium is the most abundant divalent cation and has a moderate charge density compare to Na + and Mg 2+ . Because of its large radius and coordination distance with ligands, calcium usually isn't compatible with phosphoryl transfer reactions [START_REF] Yang | Making and Breaking Nucleic Acids: Two-Mg2+-Ion Catalysis and Substrate Specificity[END_REF]. Exceptionally, the presence of calcium alone is indeed able to catalyze amino acid activation with the RNA 5'end triphosphate under an acidic pH condition by ribozyme KK13 [START_REF] Kumar | RNA-catalyzed amino acid activation[END_REF]. In another case, it also has been reported that Ca 2+ may efficiently inhibit the first splicing step of group II intron [START_REF] Erat | Divalent metal ions tune the self-splicing reaction of the yeast mitochondrial group II intron Sc.ai5??[END_REF]. Perrota and coworkers have observed an interesting reactivity switch between genomic and antigenomic HDV ribozyme by changing the preference for Mg 2+ and Ca 2+ respectively, which means the genomic HDV ribozyme structure cleaves a bit faster in Mg 2+ than in Ca 2+ while the antigenomic form acts in a contrary way [START_REF] Perrotta | A single nucleotide linked to a switch in metal ion reactivity preference in the HDV ribozymes[END_REF]). These various properties of calcium indicate that it must have been played a critical role during the evolution. A reasonable concentration of Ca 2+ was included in the cocktail buffer to stabilize the ribozyme structure and possibly play a role in the activation of the amino acids.

Another well-studied divalent cation is manganese (Mn 2+ ). This ion is the most similar to Mg 2+ (Mn 2+ radius is 0.70Å). Experiments of Mn 2+ /Mg 2+ replacement are often conducted to understand the catalytic mechanism of ribozymes. Sometimes the effects could be dramatic [START_REF] Young | Metal Ions Play a Passive Role in the Hairpin Ribozyme Catalysed Reaction[END_REF][START_REF] Pontius | Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism[END_REF][START_REF] Schnabl | Controlling ribozyme activity by metal ions[END_REF]. The smallest and simplest self-cleavage ribozyme was discovered as a manganese-dependent RNA whose activity is not affected by the presence of magnesium [START_REF] Dange | A Mn2(+)-dependent ribozyme[END_REF]). The very similar small ribozyme present in the 3'-UTR of Vg1 mRNA and β-Actin mRNA is also manganese-dependent, and

Mn 2+ can only replaceable by cadmium [START_REF] Kolev | A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA[END_REF]). In the case of the hammerhead ribozyme, the substitution of Mg 2+ to Mn 2+ dramatically promotes catalysis up to 400 fold (Roychowdhury-Saha and Burke 2006) and also exceptionally facilitate folding compared with other alkaline earth ions [START_REF] Boots | Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme[END_REF]. It may participate in stabilizing the ribozyme folding and forms tighter ions binding site that can enhance the loop-loop interactions [START_REF] Kisseleva | Binding of manganese ( II ) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy[END_REF]. At the experimental level, Mn 2+ is usually at a concentration of millimolar range. Considering the presence of Mg 2+ and Ca 2+ , we choose to limit the concentration of Mn 2+ to a micro-molar concentration.

Substrates for ribozyme aminoacylation: amino acids and activating cofactors

Considering the two chemical steps leading to the aminoacylation of an RNA (eqs. 1 and 2), the activation of the carboxyl group of the amino acids (eq. 1) represents a major barrier to overcome. In the modern genetic system, this activation is achieved with ATP ATP + aa ↔ aa-AMP + PPi………………………………... Using these leaving groups, the activated forms of the amino acids are all produced by organic synthesis [START_REF] Zhang | Peptide bond formation by in vitro selected ribozymes[END_REF][START_REF] Murakami | Aminoacyl-tRNA synthesis by a resin-immobilized ribozyme[END_REF][START_REF] Li | Ribozyme-catalyzed aminoacylation from CoA thioesters[END_REF][START_REF] Niwa | A flexizyme that selectively charges amino acids activated by a water-friendly leaving group[END_REF][START_REF] Morimoto | Flexizymes: their evolutionary history and the origin of catalytic function[END_REF].

The in vivo activation of carboxyl group is usually achieved by either of three intermediates: Because RNA does not have positively charged residues, it is not clear how a ribozyme could achieve this activation; the only known activating ribozyme [START_REF] Kumar | RNA-catalyzed amino acid activation[END_REF] has not been structurally characterized so far. Among possible strategies are the coordination through divalent ions and protonated cytidine residue(s).

Based on few reported ribozyme crystal structures or computative structures, ribozyme can fold into complex tertiary structure motif with loops, stems, bulges, junctions, twisted bases to support multiple binding sites of ligands, such as ATP, AMP, amino acid, Adenine, NAD, CoA, etc. [START_REF] Chen | Ribozyme catalysis of metabolism in the RNA world[END_REF]). ATP could have played an important role in ribozyme catalysis metabolism. If a ribozyme could catalyze aminoacylation, it firstly must provide appropriate binding pockets, such as amino acids, ATP and/or ions. In vitro selection has already illustrated such possibilities. Known ATP aptamers can either bind the adenine group [START_REF] Dieckmann | Solution structure of an ATP-binding RNA aptamer reveals a novel fold[END_REF] or strongly recognize the triphosphate group [START_REF] Sazani | A small aptamer with strong and specific recognition of the triphosphate of ATP[END_REF]). Their binding mechanism are all based on hydrogen bonds and RNA's folded structure [START_REF] Tang | Mechanism for allosteric inhibition of an ATP-sensitive ribozyme[END_REF]. Another natural selected cofactor GTP is an essential substrate for group I intron in vivo.

In vitro experiments have shown that if GTP is replaced by ATP, the first step splicing reaction can be inhibited, which indicates the GTP binding specificity [START_REF] Raghavan | A Unique Group I Intron in Coxiella burnetii Is a Natural Splice Mutant[END_REF]). Compare with ATP, the guanidine group can provide one more hydrogen bond binding site and increase the affinity to RNA molecule. In our experiment we chose both ATP and GTP as activation cofactors with 2 mM as the final concentration.

Yarus and co-workers have analyzed the properties of various RNA-bound amino acids from riboswitches, aptamers and RNPs [START_REF] Yarus | RNA-amino acid binding: a stereochemical era for the genetic code[END_REF]. Different charges and sizes of side chain could guide to different binding mechanism and efficiencies. A few base mutations can change the structure of binding motifs and make them recognize different amino acids [START_REF] Famulok | Molecular Recognition of Amino Acids by RNA-Aptamers: An L-Citrulline Binding RNA Motif and Its Evolution into an L-Arginine Binder[END_REF][START_REF] Geiger | RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity[END_REF]. Moreover, amino acids themselves as cofactors could potentially participate in the catalysis [START_REF] Roth | An amino acid as a cofactor for a catalytic polynucleotide[END_REF]. In order to offer a maximum of possibilities to the selection, we choose to work with a mixture of 19 different amino acids during the incubation step.

Other cofactors

Short polymers accumulation may have helped macromolecules to gradually construct the prebiotic world [START_REF] Pressman | The RNA World as a Model System to Study the Origin of Life[END_REF]. Besides RNA, other polymer chains, such as TNA, PNA, GNA and PDB, were also thought to be part of the original environment [START_REF] Joyce | The antiquity of RNA-based evolution[END_REF][START_REF] Yakhnin | A model for the origin of life through rearrangements among prebiotic phosphodiester polymers[END_REF]. All these hypothetical polymers could also have undergo a co-evolution process [START_REF] Higgs | The RNA World: molecular cooperation at the origins of life[END_REF].

Some of these polymers are known to affect the activity of RNA molecules [START_REF] Stolze | Novel spermineamino acid conjugates and basic tripeptides enhance cleavage of the hairpin ribozyme at low magnesium ion concentration[END_REF]). In the cellular environment, RNA is mostly present in the cytoplasm, where up to 30% in volume is occupied by different macromolecules and crowders. In vitro experiments have shown that uncharged polymers such as polyethylene glycol (PEG), can either stabilize folded RNA structures or in another case decrease the stability of hammerhead ribozyme stem helices [START_REF] Nakano | Facilitation of RNA enzyme activity in the molecular crowding media of cosolutes[END_REF][START_REF] Kilburn | Crowders perturb the entropy of RNA energy landscapes to favor folding[END_REF]. Depending on PEG molecular weight and ion concentration, the hammerhead ribozyme can exhibit different activities due to a change in cation binding properties [START_REF] Karimata | The roles of cosolutes on the hammerhead ribozyme activity[END_REF][START_REF] Nakano | Molecular crowding effect on metal ion binding properties of the hammerhead ribozyme[END_REF]). On the other hand, charged polymers like peptides with functional side-chains could also facilitate the activity of RNA in catalysis. These polymers could possibly protect RNA from degradation, promote loops closures and even strengthen double-helices (Figure 2-7 A) (van der Gulik and Speijer 2015;

Carter 2015). Recently, using group II intron as a model to mimic the in vivo condition, Firorini and co-workers have found that crowding environment can affect RNA folding and activity [START_REF] Fiorini | Mimicking the in vivo Environment--The Effect of Crowding on RNA and Biomacromolecular Folding and Activity[END_REF]. Molecular crowding was also found to help a mutated ribozyme to overcome destabilization [START_REF] Lee | Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme[END_REF].

Peptides have already been used in ribozyme selection to promote the aminoacylation. The RNA world hypothesis does not imply that RNA could perform any primordial task without cofactors such as small peptides, and both ribozymes and cofactors may have co-evolved in the prebiotic world [START_REF] Bashan | Ribosome crystallography: catalysis and evolution of peptide-bond formation, nascent chain elongation and its co-translational folding[END_REF][START_REF] Bowman | The Ribosome Challenge to the RNA World[END_REF].

As

Physico-chemical conditions: pH, incubation time and temperature

The choice of an appropriate pH is certainly critical to the success of our experiments. Although most enzymatic reactions occur in neutral or slightly alkaline pH, it is no clear what were the prebiotic pH conditions. Since molecular self-assembling started billion years ago under an atmosphere enriched in CO2, ocean were likely acidic (pH 3.5-6), and natural selection could possibly begin in this environment, where RNA, NTPs, peptide as well as some biological micro molecule are rather stable [START_REF] Bernhardt | Primordial soup or vinaigrette: did the RNA world evolve at acidic pH?[END_REF].

Ribozyme in vitro selection has been mostly operated under neutral or slightly acidic pH.

However, the only reported ribozyme KK13 that is able to catalyze amino acid activation best functions at pH 4 [START_REF] Kumar | RNA-catalyzed amino acid activation[END_REF], while the catalysis of transesterification by ribozymes is optimal at a pH around 7.0 [START_REF] Chumachenko | Rapid and Simple Ribozymic Aminoacylation Using Three Conserved Nucleotides[END_REF]). On one hand, tRNA and most of the ribozyme catalyzed aminoacylation requires slightly alkaline pH for their functions;

on the other hand, acidic pH can prolong activated amino acid half-life and prevent aminoacyl ester bond hydrolysis [START_REF] Ninomiya | In situ chemical aminoacylation with amino acid thioesters linked to a peptide nucleic acid[END_REF]). In order to best cope with these contradictory requirements, we implemented switch-pH conditions in our experimental design. The final concentration of the amino acids mixture was set to 17 mM in our experiments. Without any pH adjustment, the pH of the incubation buffer would be around 1-2. By using NaOH to carefully increase the pH to 3.5 and incubate overnight on ice, any potential amino acid activation would be stabilized under these conditions. Then, the pH was increased to 7.0 by the addition of an appropriate volume of HEPES buffer, and the solution was further incubated on ice for 2 hours to accumulate any possible aminoacylation events. The fully established protocol is shown in [START_REF] Hughes | the mechanism of the oxidation of glucose by periodate[END_REF][START_REF] Loring | Periodate Oxidation of Sugar Phosphates in Neutral Solution. I. D-Ribose 5-Phosphate1[END_REF]. When this oxidation is followed by an incubation at alkaline pH (≥ 9.5), it leads to a so-called β-elimination: the terminal oxidized nucleotide of an RNA gets removed from the chain, and yields a terminal phosphorothioate group (Figure 23456789) [START_REF] Alefelder | Incorporation of terminal phosphorothioates into oligonucleotides[END_REF][START_REF] Akbergenov | Molecular characterization of geminivirus-derived small RNAs in different plant species[END_REF][START_REF] Kawano | Novel small noncoding RNAs in mouse spermatozoa, zygotes and early embryos[END_REF].

The OD-DO Assay

The 2) On the contrary, deacylation followed by oxidation (DO) is a negative control to evaluate the efficiency of a complete elimination of the RNA from the selection procedure.

3) A positive control: Deacylation only (D).

4) An additional positive control: No Treatment (NT). All our oxidation experiments are performed in the dark to prevent the photochemical decomposition of periodate and periodate oxidation of formic acid by ultraviolet light [START_REF] Marinetti | The periodate oxidation of ribose-5-phosphate in acid and alkaline solution[END_REF]. After oxidation, the excess of unreacted periodate is usually quenched with two equivalents of glucose to redox the leftover NaIO4, and then a gel spin column is applied to remove most of the remaining NaIO3 [START_REF] Dittmar K A, Sørensen M A | Selective charging of tRNA isoacceptors induced by amino-acid starvation[END_REF]. Because the small size of our RNA molecules is incompatible with these columns, this column purification step is not implemented in our experiment. Instead, we supply 10 mM of DTT to redox NaIO3 to NaI [START_REF] Hughes | the mechanism of the oxidation of glucose by periodate[END_REF][START_REF] Ivery | The inactivation of thymidylate synthase by periodate[END_REF]. A final ethanol precipitation efficiently eliminates the oxidant.

The deacylation conditions also need to be carefully established. Most RNA molecules are unstable in alkaline pH solutions, especially when cations such as Mg 2+ , Zn 2+ , Ca 2+ are present.

In a pH higher than 9, RNA will start to degrade [START_REF] Kawano | Novel small noncoding RNAs in mouse spermatozoa, zygotes and early embryos[END_REF]). On the other hand, if the pH is not high enough, deacylation is inefficient, especially in a buffered solution without ions.

We have investigated several conditions and time-course experiments while varying pH, temperature, incubation time, and PCR cycles, (data not show) to optimize the deacylation condition.

Positive control

In order to validate the oxidation and deacylation procedures, our initial tests were realized with several chemically synthesized tRNA fragments of about 50 nt (we call them mini-tRNA) from

Staphylococcus aureus (HG003) tRNA Ala , tRNA Val , and tRNA Gly . The 3'-end of these RNA is not aminoacylated, and should thus be oxidized. All our initial oxidation tests failed to prevent the appearance of a final PCR product (30 PCR cycles are usually performed). NaIO4 cannot efficiently oxidize these RNA, and the adapter ligation still works sufficiently enough to enable RT-PCR. We suspected that the chemically synthesized RNA may still keep some protecting group at the 2' or 3'-end of the RNA that could prevent oxidation [START_REF] Caruthers | The chemical synthesis of DNA/RNA: our gift to science[END_REF]. We therefore tried our oxidation experiments with in vitro transcribed mini-tRNA Val . Figure 2-13 shows that these RNAs respond much better to oxidation. With 20 cycles of PCR, a significant difference is observed between OD and DO experiments.

To confirm the sensitivity of this protocol, it was further applied on total RNA extracted from E. 2.5 RNA ligation

Adapter design

After the OD treatment, only selected RNA that keep normal cis-diol group at the 3'-end could be ligated to a 5' phosphorylated RNA adapter with a T4 RNA ligase 1 (Rnl1). Nine different RNA ligation plays an important role in this protocol. Adapters are working as fishing reagent to "harvest" the interesting target out of the mixture. RNA ligation, especially by Rnl1, is an enzymatic reaction with low efficiency, with observed ligation rates spanning from 0 to 80% [START_REF] Turunen | RNA Ligation[END_REF]. Ligation experiments with very short (1 to 5 nt) RNA fragments have shown the sequence ApApA to be a much better acceptor substrate than UpUpU. Moreover, ApA is less active than ApApA (Kafumann and Kallenbach 1975;[START_REF] Sugino | Bacteriophage T4 RNA ligase. Reaction intermediates and interaction of substrates[END_REF]. It has also been shown that short poly A acceptors and poly U donors form a pair with good ligation efficiency [START_REF] Ohtsuka | Joining of ribooligonucleotides with T4 RNA ligase and identification of the oligonucleotide-adenylate intermediate[END_REF]. Extensive ligation experiments with random 21-nt RNA molecules have revealed that less than three un-structured nucleotides at the 3'-end and RNA are likely to be poorly ligated [START_REF] Zhuang | Structural bias in T4 RNA ligase-mediated 3'adapter ligation[END_REF]). Furthermore, a 3'-end adenosine is preferred over cytidine and guanosine for ligation, while a uridine residue is a relatively poor acceptor substrate (A > C ≥ G > U) [START_REF] England | Enzymatic oligoribonucleotide synthesis with T4 RNA ligase[END_REF][START_REF] Romaniuk | The effect of acceptor oligoribonucleotide sequence on the T4 RNA ligase reaction[END_REF]. Our own tests with random pools designed with constant 3'-end showed that a much higher ligation efficiency is observed with "UpCpA" compared with "UpCpG" ending (Section 2.2.1). On the donor side, experiments with minimal donor substrates showed a decreasing ligation efficiency with the order pCp > pUp ≈ pAp > pGp [START_REF] Romaniuk | The effect of acceptor oligoribonucleotide sequence on the T4 RNA ligase reaction[END_REF]. All the tested adapters we designed have a short 5'-end poly A/U tail with a terminal phosphate group.

Adapter J, K, L, XL and M

Adapters K, L and M (Table 2345678) were the first to be investigated. They are all single stranded, with a short poly-U tail at the 5'-end. Adapter K was originally designed to test RNA ligation with an in vitro transcribed tRNA Val from Staphylococcus auras. Since tRNA are usually well folded molecules with a short unstructured CCA ending, ligation with a single strand RNA adapter works quite efficiently based on our experiment. Intermolecular ligation between adapters is however still observed in that case. Adapters L and M were designed to solve this problem; they contain an inverted dT oligo at the 3'-end to prevent intermolecular ligation.

Although this setup was found to work well with specific tRNAs, it turns out that side-products are always present while working with pools of random RNA. Clone sequencing revealed that these short PCR side-products are primer dimers (primer of the adapter + T7 promoter primer) with a small stretch of bases in between, indicating that unspecific priming occurs on the random region, either during reverse transcription or during PCR (Figure 2345678910111213141516).

In an attempt to avoid this unspecific product, we extended the length of RNA adapter (Figure 2-16, light green) and the DNA anchor (Figure 2-16, light orange) up to 32 nt (we called it Adapter XL). With an annealing temperature of 72°C, we hoped that while using a two steps hot-start PCR procedure, this would prevent the unspecific amplifications, but it did not work. A control experiment showed that unspecific priming was a concern especially during reverse transcription: while performing an RT-PCR with RNA, RNA adapter and primer of the adapter without prior RNA ligation, the same primer-dimer byproducts were generated.

Because the RNA adapter is in excessed during ligation compared to the pool of random RNA, so is the primer of the RNA adapter during reverse transcription. Thus, unwanted associations between this primer and the random RNA region would always occur, either with or without mismatch(es) (Figure 2-17). Because these reverse transcription events are not limited by RNA ligation (as it occurs with our targeted selection), they become the dominant product of the final PCR. This issue was solved with the design of combined primer of the adapter molecules (see next Section).

Adapter Hyb-XL

Combined DNA/RNA hybrid adapters have been used as a molecular probes in some other sequencing protocol [START_REF] Dittmar K A, Sørensen M A | Selective charging of tRNA isoacceptors induced by amino-acid starvation[END_REF][START_REF] Zheng | Efficient and quantitative high-throughput transfer RNA sequencing[END_REF]. The main purpose of this design is that it may avoid any unspecific priming during the reverse transcription step. In order to work well, this strategy requires the hybrid double stranded adapter to be properly folded before ligation. It is achieved with a denaturation-renaturation procedure at low concentration. Adapter Hyb-XL is constituted by an RNA adapter linked to a DNA primer by a poly-dT bridge (Table 23456789). Once folded, this chimeric oligo has its primer base-paired to the RNA segment. Improper refolding could generate duplexes of these adapters (Figure 2-18), a structure that is not expected to affect RNA ligation and RT-PCR. An unwanted side effect however occurred with this Adapter Hyb-XL after reverse transcription. As a part of our initial design, we attempted to ligate a forward DNA anchor to the 3'-end of the cDNA (see Section 2.7), required to achieve a final PCR with essentially random pools (ref. ). The DNA anchor had a 5'-dGdG-3' sticky overhang to catch the terminal 3'-dCdC end of the cDNA. Coincidentally, the restriction site (Table 2-9, grey) of this adapter contains a 5'-rGrG-3' (red) sequence, corresponding to a 3'-dCdC-5' on the cDNA.

Because some degradation of the single stranded part of the adapter could not be prevented, this 3'-dCdC-5' became available for ligation with the DNA anchor "5'-dGdG-3'" sticky end (Figure 2-21). Sequencing results allowed us to identify the primer dimer as well as this unexpected ligation product. This design was consequently abandoned.

Adapter N and NR

In another attempt to prevent to the appearance of PCR by-product, the class III adapters N and NR with a 3' overhang were investigated. Their sticky ends with 3 or 4 overhanging nucleotides were designed to bind the complementary 3'-end of targeted RNA molecules (Table 2-10, in red). A DNA ligase was used instead of T4 RNA ligase since the bound molecules form a duplex [START_REF] Moore | Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites[END_REF]. A similar strategy was already applied to target tRNA with a TGG overhang [START_REF] Dittmar K A, Sørensen M A | Selective charging of tRNA isoacceptors induced by amino-acid starvation[END_REF]. It turns out that another side-effect occurred with this design: the unstructured DNA tail of the adapter being free, unspecific priming was detected again that occurred during the reverse transcription step (Figure 2-22). An RT-PCR control performed while incubating the adapter together with a random RNA pool without DNA ligation would also yield the primer-dimer.

Adapter O and P

Adapter O and P are the last two adapters we tested for RNA ligation, and are part of the final design of the selection protocol. These two adapters mostly overcome the problems encountered with the previous adapters: They can be efficiently ligated and enable specific RT-PCR. They differ in the restriction site (Table 234567891011), and it turns out that only Adapter P could be efficiently cleaved after the final PCR of each selection cycle, which is required before a new RNA pool can be regenerated.

Adapter O was designed with a "BmrI" recognition site. This enzyme is a Type IIS restriction endonuclease that recognizes the asymmetric 6-bp sequence ACTGGG and cleaves 5 and 4 base pairs downstream on the up and down strand respectively [5'...ACTGGG(N)5 /(N)4…3'] [START_REF] Chan | Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities[END_REF][START_REF] Bao | Expression and Purification of BmrI Restriction Endonuclease and Its N-terminal Cleavage Domain Variants[END_REF].

RNA ligation buffer

Macromolecular crowders, such as polyethylene glycol (PEG), polyvinyl alcohol (PVA)

or polyvinyl pyrrolidine (PVP), have been shown to improve intermolecular ligation by increasing the relative concentration of both donor and acceptor ends through macromolecular crowding [START_REF] Harrison | Polymer-stimulated ligation: enhanced ligation of oligo-and polynucleotides by T4 RNA ligase in polymer solutions[END_REF][START_REF] Munafó | Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA[END_REF]. Besides, the ratio of accepter versus donner is also a critical parameter for RNA ligation. Quantitative ligation can be observed at low RNA accepter, with 15-20 times excess of donner molecules [START_REF] Chumachenko | Rapid and Simple Ribozymic Aminoacylation Using Three Conserved Nucleotides[END_REF]). In our experiments, we also observed that a high concentration of RNA acceptor (up to 1 μM) strongly inhibits the ligation reaction.

In summary, RNA ligation with Rnl1 (ThermoFisher) should be performed at low concentration of RNA acceptor with an excessed of RNA donor. A crowed environment and low temperature will further improve ligation. Strong secondary structure as well as intermolecular aggregation will reduce the accessibility of the RNA termini, thus inhibiting the ligation reaction.

In order to avoid this, the ratio of selected RNA and adapter is around 1/5 to 1/10 depending on the experiment.

DNA ligation of a forward adapter: mission impossible

We initially wanted to keep the 5'-end of the RNA molecules essentially random, with the exception of a terminal 5' rGrG, required for T7 RNA synthesis (Figure 2 All our constructs were tested for DNA ligation with a T4 DNA ligase (ThermoFisher). The product(s) of ligation were amplified by PCR and analyze on agarose gel.

No substantial PCR product could be obtained with our initial construct (Class I), even with overnight ligation at low temperature. Further attempts with our class II and III DNA anchors showed that the yield of ligation could be improved, however at the expense of specificity: it was not possible to prevent the appearance of side-product during the final PCR (data not shown).

Figure 2-28: the correlation between temperature and relative activity of Taq DNA polymerase [START_REF] Chien | Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus[END_REF]).

We also tried another approach: instead of relying on a DNA ligase to attach the forward A pioneering research on Taq DNA polymerase [START_REF] Chien | Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus[END_REF] has shown that at temperatures below the optimum, like 40 to 60ºC, this polymerase still has a considerable activity. This is the reason why researchers prefer to use hot-start DNA polymerases for highfidelity PCR. Our annealing-elongation method includes two main events (Figure 2-29): 1)

Single strand DNA anchor and target cDNA are denatured at 95ºC and gradually cooled down to 30ºC in 2 hours, which allows the two oligos to anneal by both specific base-paring and inosine wobble base-paring. Notably, the 3'-"CC" end of target cDNA could only base pair with the opposite T7 promoter strand 3'-"GG" end, while the design of Adapter P doesn't allow the in excessed primer of RNA adapter to bind this DNA anchor. 2) Meanwhile, the Taq DNA polymerase was expected to slowly elongate the 3'-end of target cDNA using DNA anchor as template. After 2 hours of incubation, a reverse primer was added during denaturing, and a regular PCR program would go on from there. This strategy worked with a mini-tRNA (Figure 2-29), however with very low efficiency. Because we always experienced lower efficiencies with random pools, this approach was not pursued. In the end, we abandoned the idea of working with RNA pool completely randomized at the 5'-end. Instead, we decided to compromise and designed the shortest possible constant track the 5'-end that would allow direct PCR after the reverse transcription step (Section 2.7).

Reverse transcription

Reverse transcription (RT) is a reaction efficiency of which has been improved by engineered enzymes such as SuperScript ® III (SSIII). With our initial design requiring forward adaptor ligation (see above), we however identified a critical issue related to terminal transferase activity and template-switching activity, known to occur with RT enzymes derived from Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) [START_REF] Chen | Reverse transcriptase adds nontemplated nucleotides to cDNAs during 5'-RACE and primer extension[END_REF], SSIII belongs to this family of enzymes. It was already suspected to have this behavior, and add extra non-templated nucleotides at the cDNA's 3'-end [START_REF] Kulpa | Determination of the site of rst strand transfer during Moloney murine leukemia virus reverse transcription and identi cation of strand transfer-associated reverse transcriptase errors[END_REF][START_REF] Oz-Gleenberg | Reverse transcriptases can clamp together nucleic acids strands with two complementary bases at their 3???-termini for initiating DNA synthesis[END_REF], 2012;Zajac et al. 2013).

PCR optimization

A successful PCR amplification reveals if all steps in a selection cycle go well, in which case a pool can be regenerated for the next selection cycle. Considering the difficulties encountered with (almost entire) random RNA pools, that require the ligation of a 5' anchor, we decided to abandon this strategy. Instead, we designed the shortest possible constant track at the 5' end of these RNA that could allow the overhang of a T7 promoter to prime the corresponding cDNA during final PCR. The only difficulty is to be able to perform an efficient amplification while using the extremely short conserved region. An optimal PCR requires a melting temperature above 45ºC and below 72ºC in a classic thermocycler reaction [START_REF] Hyndman | PCR Protocols[END_REF]. Previous investigations have shown that short primers around 10 nt can achieve reliable amplification. Furthermore, it has been reported that chemical compounds like the tripeptide 1,2-dihydro-(3H)-pyrrolo-[3,2-e] indole-7-carboxylate (CDPI3), can bind to the minor groove of DNA with high affinity and allow primers as short as 8 to 10-mers to perform specific and efficient amplification [START_REF] Afonina | Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder[END_REF]. GC rich primer as short as 7-mers can be used in PCR by simply reducing the annealing temperature down to 35ºC with however much less specificity [START_REF] Vincent | Oligonucleotides As Short As 7-Mers Can Be Used for Pcr Amplification[END_REF]). Minimal primer with only 6-mers can also be used to amplify the plasmid carrying some specific restriction sites [START_REF] Ryu | Restriction primers as short as 6-mers for PCR amplification of bacterial and plant genomic DNA and plant viral RNA[END_REF]. Based on these data, we designed a series of RNA pools characterized by 7 constant positions at the 5' end and 3 specified nucleotides at the 3' end. A forward primer carrying a T7

promoter with a 7-nt overhang was designed to prime with the cDNA's 3'-end as shown in

The final SELEX strategy

All the refinements elaborated throughout the assessment of each step of our SELEX strategy could finally be combined into a working protocol (Figure 2-36). Compared with the initial design (Figure 2-1), the major change implemented in the final design is the absence of a DNA ligation step after reverse transcription. This step was investigated for almost two years, and proved to be impossible. It could be remove at the expense of the presence of a 7-nt constant track at the 5' end of the RNA pools, implying that these pools would still impose some constraints on the kind of ribozyme that could be isolated.

Results and discussion

SELEX experiment

Altogether, four different RNA libraries corresponding to the optimal design of the pools (Table 2- 3-1).

GGCACGACUCCUACACCAUAGAAGCGUUAAGCGUCA

. (((((.((.(((.....))).)))))...)).... GGCACGAAACCACACCAAACUUGCAGUAACCAAAUCA .(((.................)))............. GGCACGACGAUCAACUACAGCUUGCUGCGCCGCAUCA (((.((.(((.(.......).))).)).)))...... GGCACGACGCAUCGAUGCCCGUCGCUCUCACAAGUCA (((((((....))).)))).................. It can however be noted that despite the use of reverse primers with TGA 3' overhangs (specific to the 3' ends of the pools) the size of the clones already experienced some fluctuations after the first cycle.

The obtained PCR products were re-amplified using the AccuPrime™ Taq polymerase (Invitrogen), with which very clean products could be obtained (Figure 3 The digested PCR products were directly used for T7 transcription without further purification. Since the EarI digestion could not reach completion, a small fraction of nondigested PCR products carrying the RNA adapter sequence were still present, and could be observed during the next round RNA synthesis (Figure 3-2). This long RNA transcript did not seemingly affect the next rounds of the selection experiment. Following the experimental strategy summarized in Figure 2-36, all four RNA libraries went through successive OD treatment and re-amplification for the remaining cycles. From the 2 nd cycle, cDNA libraries became much easier to regenerate and less PCR cycles were required after the reverse transcription step. After three selection cycles, we noticed that our standard DNase treatment procedure could not eliminate all DNA after T7 transcription. Since this could critically impair the selection process, we decided to use HPLC to purify the RNA transcripts for the remaining cycles (see Supporting information HPLC purification). Since a SELEX procedure reduces the sequence diversity after each cycle, the amount of RNA used in the experiments was decreased from 0.025 nmol (cycles 1 to 3) to 0.01 nmol (from cycle 4). The related parameters are indicated in Table 3-3. After only four cycles of selection, the N23 pool became highly homogenous. This phenomenon was noticed during a bioanalyzer analysis (Figure 3-3): unlike specific RNA molecules, a random RNA pool normally shows a much boarder peak on the bioanalyzer panel due to the distribution of the secondary structures and length heterogeneity. We decided to pause the selection for this pool and to continue working with the other libraries. Figure 3-3 shows the bioanalyzer traces of the initial pools and pools of RNA observed at the final cycle. We observed that the peaks of all libraries (except for N23) became much sharper after 6 or 7 rounds of selection. This behavior suggested to us that the selection process had converged.

Consequently, we decided to proceed with sequence analysis rather than to continue with more cycles.

Sequence analysis after the final round of selection

After the final cycle, the PCR products of each four libraries were inserted into pJET plasmids using the CloneJET PCR Cloning kit. Single clones were sent for sequencing, and a total number 156 sequences were obtained. We first discuss the results obtained with each of the four libraries. An overall analysis in which these pools are compared to each other is presented afterwards.

Library N19

After 6 rounds of selection, 46 independent sequences were collected from library N19 (Table 34). The length distribution is consistent with the expected size: 50% of the sequences still have the initial size of 19 nt, while the remaining sequences are slightly longer or shorter. It can already be noticed that an appreciable fraction has a size of 23 nt. In terms of diversity, only around 40% of the sequences are unique, which means the SELEX procedure eliminated about 60% of the diversity of the pool. 5 GGCACGAACAAUGAUGUCA 1

(((((.......).)))).

6 GGCACGAACAUUGAUGUCA 1

(((((((...))).)))).

7

GGCACGAACACUCUUGUCA 1

((((.((....)).)))).

8 GGCACGAACAUCGACGUCA 1

(((.(((...)))..))).

22

GGCACGAAGCUACUCGUCGGUCA 1

(((((((......))))..))).

26nt

GGCACGAUUAUUAGUCAACUAACUCA 1 (((..........)))..........

An examination of these sequences reveals that they share some similarities (Table 34).

In particular, many of them bear a same ACA motif that follows the 5' GGCACGA constant track (Table 3-4, underlined). This motif is most often found inside a loop in the predicted secondary structures, suggesting that it could have a structural or functional role. These secondary structures also exhibit some similarities. Most of them fold into a stem-loop with a single-stranded 3' end. However, many of these free 3' ends only consist of the terminal "A" residue that is not expected to be flexible enough to reach any potential catalytic site (see [START_REF] Lehmann | Efficiency of a self-aminoacylating ribozyme: effect of the length and base-composition of its 3' extension[END_REF]. It does suggest that this kind of species is probably a false positive result.

The secondary structure of these RNA may assist them to avoid periodate oxidation and/or allow a much more efficient RNA ligation. For the longest selected RNA, the free single-stranded 3'-end is usually much longer (Figure 3-4, B), a structure that would be consistent with a catalytic activity: similar to the tiny Yarus ribozyme [START_REF] Illangasekare | Specific, rapid synthesis of Phe-RNA by RNA[END_REF][START_REF] Lehmann | Efficiency of a self-aminoacylating ribozyme: effect of the length and base-composition of its 3' extension[END_REF], the free 3'-end could reach a plausible binding pocket constituted by the large predicted loop. This "Hammer Throw" shape secondary structure is commonly found among those sequences; it includes a 7 to 11-nt loop, a three base-pair stem and a single stranded 3'-end tail of different lengths.

Library N23

The selection of library N23 was stopped after the 4 th round because the bioanalyzer trace showed a very sharp peak (Figure 3-3), that we initially suspected to be due to a sequence contamination, or an accident that created a massive loss of sequence diversity. A small sample was sent for sequencing, from which 13 independent sequences were obtained (Table 345).

Interestingly, 11 out of 13 sequences still keep the correct size of 23 nt. These sequences are almost all different, but 2 out of 13 sequences (Table 3-5, No.1) are identical to 2 sequences found in library N19. Since all the experiments were performed independently, we are very confident that is not a cross contamination. In terms of secondary structure, no clear motif could be detected besides the common stem-loop structure (with either a Key" or "Hammer Throw" shape). Two sequences (No. 5 and 9) have no predicted secondary structure, suggesting that the pool was not yet converging; it seems we gave up with library N23 a bit too early. 12 GGCACGACCACGUGACCCCCCUCA 1

((((((....)))).)).......

Library N30

It is very instructive to already compare the sequencing result of library N30 (Table 3456with the two previous libraries. The length distribution is quite board, and no clone kept the initial size of 30 nt. However, among the 24 identified sequences, many of them are absolutely identical to sequences found in libraries N19 and N23, revealing that a same convergence phenomenon occurred independently, during which not only the sequence but also the size underwent a selection process. 14 36nt GGCACGACCAGCGUUCCUCUAGACCCCUACGUCUCA 4

((.(((.....))).))...((((......))))..

37nt

GGCACGACCAGCGUUCCUCUGGACCCCUACGUCUUCA 5

((.(((.....))).))...((((......))))...

In addition, the longest sequences of library N30 are also identical to the most abundant clones identifies in the N37 library (see below).

Library N37

Since the N37 library is experimentally easier to handle, we run one more selection cycle with this pool. Altogether, 9 clones were sequenced from round 6 (Table 34567), while 64 sequences were identified from round 7 (Table 345678). From the table, we can see that the diversity of the pool has dropped dramatically comparing with the first selection cycle (Table 3-2 Thus, it turned out that the "strange" behavior of the N23 pool, that seemed to prematurely converge towards certain sequences, was likely due to an "attractor effect" that also affected all three other pools.

A comparison of all libraries shows a convergence towards certain structures that occurred for all of them. A classification of these structures into seven categories is presented in Tables 3456789and 3-10. Since aminoacylated RNA are expected to have a different retention in the column of a liquid chromatography experiment (e.g. [START_REF] Lehmann | Efficiency of a self-aminoacylating ribozyme: effect of the length and base-composition of its 3' extension[END_REF]), a time delay on reverse phase 84 HPLC is expected. We decided to use this different approach to further test the activity of the pools. Practically, pools of RNA can be injected on the HPLC right after an incubation reaction.

We tested the library N23 after 4 cycles selection, N30 after 6 cycles selection and N37 after 7 cycles selection. No delay in the signals was apparent on the overlay of the two "+aa" experiment and "-aa" control (Figure 34567891011).

An interesting result was however found when we tested the N37 library: while using a similar procedure as with the N19 pool (Figure 3-10), the signals were consistent with the protection of the 3'-end of some RNA due to the presence of the amino acids, suggesting that aminoacylation did occur (Figure 3-12). This result still needs to be reproduced. Besides, we also choose a few specific sequences to test them individually with HPLC. In total 23 specific sequences were divided into 5 batches for the tests (Supporting information).

No clear signal could be detected by HPLC.

Conclusion

We established a SELEX protocol to isolate self-aminoacylating ribozymes while using optimized OD-DO assays. A carefully designed hybrid RNA adapter was used to achieve efficient RNA ligation. Moreover, this hybrid adapter can significantly prevent unspecific priming during the reverse transcription step. In this protocol, we also used primers with small overhangs to minimize RNA size heterogeneity during the selection, and to avoid an unmanageable step (the ligation of a forward adaptor). Combining these techniques, we could limit the size of the conserved regions down to 10 nt.

The available substrates (a mixture of 19 amino acids with ATP/GTP cofactors) required the targeted ribozymes to activate the amino acids with either ATP or GTP and realize transesterification to the 3'-end. Although we could finalize a working SELEX procedure while minimizing the constant tracks at both the 5' and 3' ends, tests with the libraries obtained after 6 to 7 cycles failed to reveal any clear activity. Furthermore, we identified an unexpected phenomenon that prevented our selection protocol to isolate the targeted ribozymes. Besides these considerations, there are several possible reasons that may have cause the failure of our approach:

1) The cocktail buffer is inappropriate for aminoacylation. It is the risk of a "blind selection" existing in all in vitro SELEX experiment. Further investigation could allow us to improve the composition of the buffer.

2) Periodate oxidation could not eliminate hundred percent of non-aminoacylated RNA.

Some of the RNA molecules, for instance the ones with strong secondary structures could possibly resist oxidation, will surely be selected.

3) Our idea was to select a class of small ribozymes. Our initial pool may not be large enough to fulfill the requirements of the reactions being investigated. Ribozyme being able to catalyze both amino acid activation and transesterification may require larger structural motifs. In order to promote nucleophilic attack, RNA molecules should be able to tightly bind amino acid and ATP substrates. and bring them to a reaction center, meanwhile the 3'-end of RNA should also be able to reach this center to accept the activated amino acid. This sophisticated binding system may require a longer RNA chain and larger initial pool of RNA to provide a higher diversity to the selection.

4) Given the unexpected phenomenon that occurred during selection, the SELEX protocol should be redesigned to prevent the possibility of these effects.

5) At last, some additional experiments should still be undertaken to verify whether some selected sequences could still promote aminoacylation.

Chapter II Identification of aminoacylated RNA from E. coli

Introduction

As part of our work on aminoacylated RNA, we wanted to develop a robust OD-DO procedure to not only investigate the possibility of new self-aminoacylating ribozymes (Chapter I), but also to analyze total RNA from different species, and find out whether aminoacylated RNA other than tRNA and tmRNA could be identified in living cells. For this purpose, we choose to work with total RNA from Escherichia coli, a well-studied Gram-negative bacterium. Our There are several indications that other types of RNA molecules could be aminoacylated in cells. In plants, it has been shown that some RNA viruses bear transfer RNA-like structures (TLSs) that require aminoacylation to achieve a full cycle of infectivity [START_REF] Dreher | Role of tRNA-like structures in controlling plant virus replication[END_REF]. Another interesting fact is the aminoacylation property of tRNA minihelixes [START_REF] Francklyn | Enzymatic aminoacylation of an eight-base-pair microhelix with histidine[END_REF]: in vitro experiments showed that small stem-loop mimicking the acceptor stem of tRNA can be aminoacylated by the aminoacyl-tRNA synthetase, although at low efficiency. It is not known whether this type of aminoacylation could also spontaneously occur in vivo, and have functional purposes.

By using the deep sequencing technology, our investigation could highlight new RNA candidate for aminoacylation, and further explore the rules that govern the aminoacylation levels of tRNAs.

Method

Our approach relies on the careful preparation of total RNA extracted from E. coli followed by oxidation and control protocols: OD, DO, D and NT (Supporting information OD-DO treatment). Similar to the SELEX experiment, OD treated sample are expected to provide the reads of aminoacylated RNA. DO processed samples is a negative control that should not provide any substantial specific signal except for RNA with 3' methylation (one unknown phenomenon in bacteria) [START_REF] Motorin | RNA nucleotide methylation[END_REF] or if DO treatment may still leave nonoxidized 3' terminations and degraded small RNA fragments during the bench manipulation. D processed samples should make any RNA available for sequencing (including aminoacylated RNA) while NT samples constitutes the untreated positive control.

Primary attempt with a standard Illumina RNA-seq protocol

In our first attempt to acquire deep-sequencing data with treated samples of total RNA, a standard RNA-seq bank preparation protocol was followed (TruSeq Small RNA kit, Illumina).

We provided treated RNA samples to the Plateforme de Sé quenç age facility at Gif-sur-Yvette treatment was performed to ensure that the 5'-end of the RNA samples had a monophosphate.

First sequencing Results

Sample preparation

Our former total RNA samples were prepared by our collaborators from ETH in Zurich (T.

Grentzinger and A. Marchais). They provided us with total RNA samples from E. coli BL21 strain extracted during exponential phase with Trizol, in a final form of frozen aliquots of 5 µl at a concentration of 3 µg/µl. They were shipped in dry ice, and immediately stored at -68°C upon receiving. An analysis with bioanalyzer however revealed that the quality of these samples was not high enough for deep sequencing. We decided to prepare new samples by ourselves from the E. coli BL21 strain kindly provided by the laboratory of Philippe Bouloc (I2BC).

Total RNA from E. coli BL21 was extracted at OD600 point: 0.24, 0.47, 1.36 as well as an overnight culture, the final sample was constituted by an equal amount of each extraction. This sample was split into four aliquots, each of them (~4 µg total RNA) being processed through either the OD, DO, D or NT protocol (Supporting information, total RNA extraction). Then, these samples were treated with DNase I, re-suspended in 15 µl DEPC-water and quantified (Table 2.1). An equal amount of spike was added in each sample (see below) and the quality of the RNA was checked with Bioanalyzer (Figure 2.2). The presence of ribosomal RNA confirmed that no excessive degradation occurred during the treatments, and the samples were sent for bank preparation at the Plateforme de Sé quenç age (Imagif, Gif-sur-Yvette).

Spikes added to the samples:

Spikes are exogenous RNAs that allow the normalization of signals obtained from a series of samples. Three spikes of a size comprised within the defined categories (10-50 nt, 50-100 nt and 100-250 nt) were added in equal amount to the four treated samples (Table 2.2).

RNAspike_25 and RNAspike_65 are chemically synthesized RNA (IDT), and correspond to initial portions of RNAspike_131. RNAspike_131 is the transcript of the control template of the MEGAshortscript™ kit (Ambion), a fragment of human 18S rRNA. This spike was in vitro synthesized and HPLC purified.

Bank preparation

The samples were gel purified at the Sequencing Platform Facility at Gif-sur-Yvette, which allowed a splitting into the three defined size categories (10-50, 50-100 and 100-250 nt).

Importantly, no RNA fragmentation was applied, a procedure that is often part of an RNA-seq bank preparation. The 12 samples resulting from this procedure were individually labelled during bank preparation by a specific index sequence on the 3' Universal Adaptor of the TruSeq Small RNA kit (Illumina) (Table 2.3). The 12 cDNA banks were processed by Beckman-Coulter (USA) for deep sequencing using the Illumina HiSeq2500 pair-end 2x125 technology.

Deep-sequencing results

On average, 20.75 million reads per bank (min = 13 Mio; max = 25 Mio) were sequenced, each transcript being constituted by R1 and R2 reads. An analysis of these transcripts (FastQC report) revealed an excellent overall quality.

Demultiplexing, adapter removal and mapping was achieved with Bowtie [START_REF] Langmead | Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[END_REF]). R1 and R2 reads were mapped on a reference genome of E. coli BL21. During this operation, we realized that a large fraction of the reads could not map on E. coli BL21 genome, and did neither match our spike sequences. A BLAST search revealed that our samples became contaminated with some material from Saccharomyces cerevisiae (chromosome XII) (Table 2.4). An investigation could not reveal the origin of this contamination. A sufficient number of E. coli reads were however available for mapping, and allowed us to perform a preliminary analysis, and compare the reads found in some selected tRNA between the four tested conditions. We first established the number of reads mapping on the spikes in order to normalize the signals observed in each condition. Although the number of reads in the 50-100 nt category was reduced due to S. cerevisiae contamination, most tRNA sequences would fall into this category.

We therefore decided to examine it in priority.

Comparison between OD, DO, D and NT signals in the 50-100 nt category

An examination of the files with the IGV software [START_REF] Robinson | Integrative genomics viewer[END_REF][START_REF] Thorvaldsdottir | Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration[END_REF]) could immediately reveal an unexpected result: it appears that the oxidation treatments did not reduce the reads coverage observed in OD and DO samples, compared with the D and NT controls. The situation observed with tRNA Met is not an exception: although there is a variation of the relative proportions of read counts between treatments, an inspection of the read coverages for other tRNA shows that this phenomenon is general. A striking feature, likely related to this undesired phenomenon, is that full-length reads are almost absent: they only constitute a few percent of the total number of reads for a given molecule (Figure 2.4). Why are there so few entire tRNA molecules in the banks? In addition to fragmentation, a plausible explanation is that modified bases present on full-length tRNA prevent the progression of the reverse transcriptase during cDNA synthesis [START_REF] Motorin | Identification of modified residues in RNAs by reverse transcription-based methods[END_REF].

This final step of bank preparation (Figure 2.1) has to be fulfilled in order for a considered read to undergo final PCR. An undesired consequence of this phenomenon is the following: because only fully modified tRNA are (essentially) aminoacylated by the aminoacyl tRNA synthetase, our bank preparation protocol could not quantitatively access the information about the level of aminoacylation of tRNAs.

We still wanted to know whether our present protocol could provide some information about tRNA aminoacylation. An inspection of the 3'-end of reads mapping tRNA genes revealed an unexpected signature of the DO reads when compared with those undergoing other treatments: instead of a regular -CCA 3' termination, the large majority of these reads lack the terminal adenosine (-CC 3' termination). that this ratio has a high dispersion for the NT control (Figure 2.6, A). Because we don't know whether an A76 removal reflects the situation with full-length tRNA or tRNA fragments (see above), any interpretation would be hazardous. While this effect needs to be further investigated, it can be mentioned that A76 removal by RNase Z has been reported in E. coli [START_REF] Ezraty | The RNase Z Homologue Encoded by Escherichia coli elaC Gene Is RNase BN[END_REF][START_REF] Takaku | Escherichia coli tRNase Z can shut down growth probably by removing amino acids from aminoacyl-tRNAs[END_REF], and could play a role in translation regulation. alaU, alaV, alaW, alaX, argQ, argU, argV, argW, argX, argY, argZ, asnT, asnU, asnV, asnW, aspT, aspU, aspV, cysT, glnU, glnV, glnW, glnX, gltT, gltU, gltV, gltW, glyT, glyU, glyV, glyW, glyX, glyY, hisR, ileT, ileU, ileV, ileX, leuP, leuQ, leuT, leuU, leuV, leuW, leuX, leuZ, lysQ, lysT, lysV, lysW, lysY, lysZ, metT, metU, metV, metW, metY, metZ, pheU, pheV, proK, proL, proM, selC, serT, serU, serV, serW, serX, thrT, thrU, thrV, thrW, trpT, tyrT, tyrU, tyrV, valT, valU, valV, valW, valX, valY, valZ] Finally, we wanted to see whether the proportions of the [-CC + -CCA] read counts between different tRNA genes could reflect the relative abundance of these tRNA observed in E. coli. The comparison of our data with the tRNA abundance experimentally determined by Kurland and coworkers using 2D high resolution gel analysis (Dong et al. 1996b) show that our data are far from being representative (Figure 2.8). An examination of the NT read counts for individual tRNA reveals that lysine and isoleucine tRNA reads are almost absent (<10 reads) when some leucine tRNA have more than 20,000 reads. Although the various effect(s) of base modification on reverse transcription efficiency is unknown, it is quite plausible that it may explain these discrepancies [START_REF] Zheng | Efficient and quantitative high-throughput transfer RNA sequencing[END_REF].

In conclusion, a standard RNA-seq protocol is not appropriate for the study of the aminoacylation level of RNA molecules due to fragmentation during bank preparation. In addition, the presence of modified bases on tRNA may not allow a quantitative assessment of these species in solution. This second issue has recently been addressed (or at least been partly overcome) with a protocol during which methylations are removed prior to bank preparation (Figure 2.9) [START_REF] Zheng | Efficient and quantitative high-throughput transfer RNA sequencing[END_REF]. In this improved procedure, the use of a template-switching polymerase (TGIRT) also enables a higher amount of cDNA resulting from aborted reverse transcription events to still be included in the bank. This approach is however problematic in our study of aminoacylation because it requires demethylase treatments in alkaline buffers with

New RNAseq protocol for the study of the aminoacylation level of RNA transcripts with modified bases

The identification of issues in the study of aminoacylated RNA species with a standard RNAseq protocol allows us to propose a new protocol optimized for that purpose. Important considerations are the following:

-In order to establish a reliable comparison between OD, DO, D and NT samples, our previous investigation showed that 3' adaptor ligation should occur immediately after these treatments while gel purification and PNK treatment steps must be avoided.

-Since we are interested in the aminoacylation level of the 3' end of these molecules, it is not necessary to obtain full-length cDNA of the RNA transcripts since 5'-end sequence information is not essential. A size of at least ~20 nt is however desirable in order to achieve proper mapping of these transcripts on the genome.

Since the reverse transcriptase has high probability to drop and generate truncated cDNA when it encounters modified bases [START_REF] Zheng | Efficient and quantitative high-throughput transfer RNA sequencing[END_REF], a usual two-step adapter ligation cannot be performed. In order to add a 5' adapter, this protocol combines reverse transcription with the RNA 5'RACE technique [START_REF] Frohman | Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer[END_REF][START_REF] Innis | PCR protocols -A guide to methods and applications[END_REF]. Full-length and truncated cDNA then undergo poly-A tailing with a Terminal deoxynucleotidyl Transferase (TdT) in conditions for which a tail of the order of 20 to 30 adenosine residues is added. This tail allows a reverse adapter comprising a tail of 17 dT at the 3' end to prime the 3' end of these cDNA. A two-step PCR is achieved with a reverse universal primer and a forward indexed primer, which finalizes bank preparation.

Final PCR amplification

The second and final amplification allowed us to add the long Illumina primers to the samples, required for deep-sequencing multiplexing. The reverse universal primers are all characterized by a different index (Table 2.6). 2.15) showed that the majority of the products had a size significantly higher than adapter dimers, suggesting that most of the constructions had an insert.

Results

The samples were sent for sequencing (Beckman -Genewiz). An issue about the quality of these samples was revealed by bioanalyzer analyzes performed by them. There was a conflict about the size of the most abundant product: when our gel analysis suggested a size of at least 150 bp (Figure 2.16), the bioanalyzer traces showed a size of 135 bp, very close to the size of adapter dimer constructions (Figure 2.17). To resolve this discrepancy, we decided to clone our samples and send colonies for sequencing. Among the "B-2" OD batch, only 4 out of 30 clones had an insert, two tRNA Leu fragments and two others unknown sequences (Figure 2.18), while the results where even worse (which is not surprised) with the "B-2" DO sample (2 out of 30 with an insert). Although this verification was disappointing, we still decided to proceed with deep sequencing, assuming that even with 10% of constructions with inserts, the amount of information would be already high enough to possibly uncover some interesting data. It turned out that sequencing did not only provide very few reads (67 million altogether, i.e. only about 4 million reads per bank); the quality of these reads (FastQC report) was also extremely poor (% of >= Q30 bases: 9.79; Mean quality score: 14.66).

The origin of the low quality of the reads was quickly identified, and pointed out a flaw in the design of our forward universal primer. The first 4-5 sequencing cycles are devoted to the identification of cDNA clusters by the sequencer, which requires the highest possible diversity of sequences. Because the sequencer starts to collect information about the clusters at the position right after the forward universal primer, all the clusters gave a unique signal corresponding to a dT residue (from the polyT17 overhang). The machine could thus not locate the precise position of these clusters, which had a dramatic effect on sequencing. The fact that the majority of the cDNA did not contain an insert also contributed to the very low overall quality of sequencing. The identification of this issue allowed us to design a new universal forward adapter that could eliminate the problem (see below).

Despite the low number of reads and the high amount of sequencing errors, we still tried to extract some information from these reads while focusing on particular tRNA. Obviously, the disadvantage of this modified PCR is to cause a globe bias that the final selected RNAs will all only have "CCA" ending. However, previous study had shown that the "CCA" ending is essential for the recognizing by aminoacyl tRNA synthetase (Figure 2.21).

Base mutations will dramatically change the aminoacylation ration of tRNA [START_REF] Zhou | Role of tRNA amino acidaccepting end in aminoacylation and its quality control[END_REF].

In fact, the known tRNA-like RNAs so far all have similar tRNA motifs and some even highly mimic tRNA's 3'-end (See Part II). Here we assume that all the in vivo aminoacylated RNA should have a "CCA" ending.

Perspective

The first results obtained with our innovative protocol are not exploitable at this stage.

However, several issues could be identified during this second attempt, and we are confident that with new samples prepared using an improved procedure, deep sequencing data will provide fully exploitable data with which exciting discoveries on tRNA aminoacylation, and possibly the discovery of new kind of aminoacylated RNA can be achieved.

Supporting Information

Experimental assay

Chapter I

DNA templates annealing T7 promoter (100 µM) 20 µl, cT7 DNA template (100 µM) 20 µl (N19 to N37 respectively), H2O 5 µl and NaCl (1 M) 5 µl; Mixture is incubated in 95ºC water bath for 5 min then cool down to room temperature overnight.

RNA adapter annealing

RNA adapter (Adapter Hyb-XL, N, NR, O, P) (100 µM) 1 µl, DEPC-water 99 µl; Heat the sample by PCR machine up to 90º C for 2 min then drop the PCR tube into liquid nitrogen immediately, then do ethanol precipitation to collect the pellet for RNA ligation.

Initial pool RNA T7 transcription and DNase treatment

Annealed DNA templates (0.1 nmol), Ambion ® T7 2X Buffer 10 μl, NTPs (10 mM) 8 µl, T7 RNA Polymerase 1 μl, H2O fills up to 20 μl; Mixture incubates at 37°C for 4 h. Add 2 µl Turbo DNase I (10 U/μl) and incubate 1h at 37°C . RNA products are purified by Phenol/Chloroform extraction.

Phenol/Chloroform extraction

Reaction solution 22 μl, NaOAc/HOAc buffer 15 μl, H2O 115 μl, Phenol/Chloroform/isoamyl alcohol (25:24:1) Mix (Sigma) pH 4-5 150 μl; Vortex 1 min and centrifuge for 10 min, then transfer the aqueous phase to a new tube for ethanol precipitation.

Ethanol precipitation

Aqueous phase 150 μl (including 15 μl NaOAc/HOAc buffer), Glycogen (ThermoFisher) 1 μl and 100% ethanol 450 μl. Incubate at -20º C for at least 45 min; Centrifuge sample for 30 min at 4º C with speed 15000 rpm then wash the pellet by 70% cold ethanol (DO NOT vortex), continuously centrifuge for another 15 min at 4º C with the same speed. Air dry the pellet and re-suspend in DEPCwater.

The 1 st cycle selection 1) Preparation

Prepare sterilized 1 M NaCl, KCl, NaOH solution, 100 mM ATP and GTP (ThermoFisher), 250 mM CaCl2, 250 μM MnCl2 solution. Dilute 1M MgCl2 (Sigma) to 250 mM. Prepare 0.01% polylysine and poly-arginine as well as 250 μM spermine (Sigma). Amino acids mix (Sigma) contains 2.5 mM Ala, Arg, Asp, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, Val and 1.25 mM Cys.

116

2) Aminoacylation buffer

Chapter II

Total RNA extraction

All the experiments have to be done with gloves, on ice and manipulated with RNase-free tips and DEPC-water. E. coli strain either BL21 (PhB101) or MG1655 (PhB1463) is from the lab collection of Philippe Bouloc. Streak the E. coli strain on LB plate and incubate at 37º C overnight to pick a single clone for pre-culture. Run LB pre-culture at 37º C overnight (180 rpm). Prepare Falcon tube with either 7ml or 15 ml 100% Ethanol at -80º C overnight. The next day, run three parallel cultures at the same time. Dilute 1 ml pre-culture into 100 ml fresh LB culture and incubate at 37ºC (180 rpm). Collect three OD600 points at 0.2, 0.4 and 0.8 culture to quench in equal volume of 15 ml cold ethanol (OD600 0.2) or 7 ml cold ethanol (OD600 0.4 and 0.8). Centrifuge Falcon tube 15 min (4000 rpm) at 4º C and re-suspend the pellet in 600 μl lysis buffer (0.02 M NaOAc/HOAc pH 5.5, 0.5% SDS, 1 mM EDTA). Mix the lysate pellet with 600 μl acid phenol and 100 μl water, vortex to mix well. Add 1/10 volume 3M NaOAc/HOAc pH 5.5 and vortex to mix well. Centrifuge 5 min at 13000 g and transfer the aqueous phase to a new tube. Repeat the Phenol extraction step. Transfer the aqueous phase into a new tube and mix with 600 μl chloroform. Centrifuge and collect the aqueous phase to do ethanol precipitation overnight. Re-suspend the total RNA pellet into 100 μl DEPCwater and aliquot in several PCR tubes storing at -80º C.

OD-DO treatment

Cover all the tubes with aluminum films; Dissolved 5.4 mg NaIO4 in 250 μl water to prepare 0.1 M sodium-periodate solution; All the oxidations are performed in dark and on ice. After each oxidation, add 10 μl Glucose (0.5 M) and equilibrate for 15 min at room temperature. Then add 6 μl DTT before precipitation. After adding the ethanol, add 1 µl RT primer (50 µM) in the solution. After precipitation, re-suspend the pellet into 13 μl DEPC-water.

Reverse transcription

RNA ligation pellet 13 μl, dNTPs mix (10 mM) 1 μl; Incubate at 70°C for 2 min then chill on ice. Add 5X RT Buffer 4 μl, DTT (100 mM) 1 μl, SuperScript III ® Reverse transcriptase (200 U/μl) 1 μl. Incubate 1 h at 55°C then chill on ice and ready to load on gel

Agarose gel purification

Prepare 3% agarose gel with ethidium bromide (2.4 g agarose and 80 ml TAE 0.5X running buffer). Load the reverse transcription products directly on gel. Run at 135 voltages for 5 min, then switch to 100 voltages for 75 min. Cut the gel from 35 bp to 200 bp.

Break the gel and recover cDNA

Prepare the gel breaker tubes by using 0.8 mm needle punch the bottom of 0.5 ml tube 3-4 times to create small holes. Cut the gel into suitable size (DO NOT chop into small pieces) and put it into the 0.5 ml gel breaker tube, then cover it with a 2 ml tube. Centrifuge at 15000 rpm for 5-10 min to make sure there is no gel left. Suspend crushed gel by 450 µl H2O and 50 µl TE buffer per tube then shake overnight at 20°C. The next day, transfer gel solution into a Corning Costar Spin-X ® filter tube then centrifuge at 15000 rpm for 15 min, collect the wash flow and precipitation.

TdT tailing

Recovered cDNA 500 ng, 5X TdT buffer 10 µl, 10 mM dATP 0.75 µl, TdT enzyme (20U/µl) (ThermoFisher) 0.5µl, H2O fills up 50 µl. Incubate at 37°C for 45 min then stop the reaction by heating up to 70°C for 10 min (10-30 A will be added

PCR products purification

Using 30K Amicon ® Ultra-0.

  Développement d'une méthode SELEX pour l'identification de ribozymes pour l'aminoacylation et analyse d'ARN aminoacylés dans le transcriptome d'Escherichia coli Les ribozymes sont des ARN naturels ou artificiels possédant une activité catalytique. Les ribozymes artificiels ont été identifiés in vitro par la méthode SELEX, et plusieurs d'entre eux ont été caractérisés par des études cinétiques. Ces molécules sont impliquées dans des réactions de clivage, de ligation, de modification d'extrémités d'ARN, de polymérisation, de phosphorylation et d'activation de groupements acyl. Parce qu'elle est nécessaire à la traduction, l'aminoacylation des ARN joue un rôle évolutif important dans la transition du monde de l'ARN vers le monde moderne de l'ADN et des protéines, et elle est centrale à l'établissement du code génétique. Plusieurs ribozymes catalysant le transfert d'acides aminés à partir de cofacteurs activants ont pu être isolés et caractérisés depuis une vingtaine d'années, ce qui a documenté la possibilité d'aminoacylation d'ARNt en l'absence des aminoacyl ARNt synthétases.

  coli. Dans ce deuxième travail, notre but est d'identifier tous les d'ARN aminoacylés par séquençage massif, avec à la clé la découverte possible de molécules autres que les ARNt et ARNtm. En utilisant les ARNt comme modèle, nous nous sommes aperçus qu'un protocole RNAseq standard n'était pas adapté à cause des bases modifiées présentes sur ces molécules. Nous avons développé et mis au point un nouveau protocole pour l'identification de n'importe quelle séquence aminoacylée en 3'. La nouvelle approche présentée devrait permettre l'étude exhaustive de l'aminoacylation de toutes les séquences présentes dans l'ARN total. Summary Development of a SELEX method to uncover auto-aminoacylating ribozymes and analysis of aminoacyl RNA from Escherichia coli transcriptomes Ribozymes are natural or in vitro selected RNA molecules possessing a catalytic activity. Artificial ribozymes have been extensively investigated by in vitro SELEX experiments, and characterized by kinetic assays. Ribozymes are involved in RNA cleavage, ligation, capping, polymerization, phosphorylation and acyl activation.

  to express my deep gratitude to my supervisor Jean Lehmann for his dedicated guidance, sympathy, generosity and supports during all four years. It is him who always encouraged and helped me to overcome the difficult moments in research. I feel extremely lucky to have chance to work with and learn from him. It is my great honor to have opportunity of working in Daniel Gautheret's lab with all my colleagues. It was also an honor to have collaboration with Philippe Bouloc, Annick Jacq and my tutor François Michel, from whom I received a lot of encouragements and advice. I am highly thankful to my colleague Florence Lorieux who helped me to accomplish the sequencing experiments, and also Coline Billerey and Marc Gabriel who have contributed to my thesis with bioinformatics analyses. I would like to thank all my wonderful and kind colleagues Claire Toffano-Nioche, Fabrice Leclerc, Nicolas Chevrollier, Chantal Bohn, Elena Disconzi, Rémy Bonnin, Claire Morvan and Aurélie Jaffrenou. They have helped me a lot in many occasions since I arrived in France. I truly appreciate all the helps from my best friends, Audrey Vingadassalon, Lê Lâm Thảo Nguyê n, and Nguyễn Ngọc Ẩn. With their whole-hearted supports and kindness, we have been through very memorable experiences together. I would also like to thank my Chinese colleagues DENG Yiqin, LIU Wenfeng, LUO Xing and LI Jia. We have been sharing lots of wonderful moments in daily life, research experiences and making progress together. I especially would like to express my gratitude to Dr. Shixin Ye-Lehmann who is always following my work, considering about me and trying the best to help me. I am grateful for being supported by Chinese Scholarship Council (CSC) [File No. 201206140111] during four years of PhD study and all the hard work from the "Education service office, Embassy of P.R. China in France".

Figure 2 .

 2 Figure 2. Candidate precursors (nucleic acid-like molecules) to nucleic acid. a, Threose nucleic acid (TNA); b, peptide nucleic acid (PNA); c, glycerol-derived nucleic-acid analogue (GNA); d, pyranosyl-RNA. "B", nucleotide base.[START_REF] Joyce | The antiquity of RNA-based evolution[END_REF]

Figure 3 .

 3 Figure 3. Common secondary structure of RNAs. Including: stem, bulge, internal loop, junction and end-loop. Nucleotides are represented by solid balls and hydrogen bonds by a line (Ádá m Kun 2015).

Figure 4 .

 4 Figure 4. The first stage of RNA world evolution.

  evolution because it allows recombination. Scientists have found many self-cleaving ribozymes either in cells or in viruses that can perform very accurate cleavage. Prominent examples include HDV ribozymes and group I/II intron. They might be ancient ribozymes that survived the transition to the modern biology world (Jimenez et al. 2015).

Figure 5 .

 5 Figure 5.(1) Random activated monomers randomly self-assembled into functional and non-functional oligonucleotides (2), which could serve as templates (3) that guide the complementary strand synthesis (4). Inter-and intramolecular recombination allows more copies of RNA transcripts to yield (5) to promote the replication and evolution (6). Being through the natural selection, RNAs could evolved to more sophisticated structures and functions (7), leading to the appearance of the first protocell and the early biocatalysts of the RNA world (8). Illustration from[START_REF] Hernandez | Chemical origins of life: Prebiotic RNA unstuck[END_REF].

Figure 6 .

 6 Figure 6. Down-hill acyl-transfer reactions in translation. Arrows 1 to 3 indicate the three status of translation: 1) Acyl-activation, 2) hydroxyl group acylation and 3) peptide bond formation. All three reactions can be catalyzed by different ribozymes generated by in vitro selection (Schimmel and Kelley 2000; Ellington et al. 2009) in different buffering conditions.

Figure 7 .

 7 Figure 7. Example of typical aptamer pool structure and classical selection process (Wilson and Szostak 1999).

Figure 8 .

 8 Figure 8. A general acid-base mechanism of RNA cleavage. A general base can deprotonate the 2' hydroxyl of the nucleophile, positioned in-line with the 5' O leaving group. Then a phosphorane transition intermediate can lead to the transesterification reaction depending on the stability of the transition status.Cleaved upstream RNA will construct a 2'-3' cyclic phosphate and release the downstream as an oxyanion nucleotide.[START_REF] Jimenez | Chemistry and Biology of Self-Cleaving Ribozymes[END_REF].

Figure 8 .

 8 Figure 8. Artificial ribozyme identified by SELEX experiments suggest the possibility of an RNA world metabolism. Most of the ribozymes require the Mg 2+ or Ca 2+ as cofactors and catalyze reaction at a range of pH value from 6.5 to 8.5. Exceptionally, amino acid activation so far has not been demonstrated at physiological pH (Kumar and Yarus 2001).

Figure 9 .

 9 Figure 9. Left: Amino acid starvation response in E. coli (Ross et al. 2013); Right: The T-box regulatory system (Henkin 2008).

Figure 10 .

 10 Figure 10. Charged and uncharged tRNA plays various roles in living cell, which are mostly link to translation (Raina and Ibba 2014).

  similar ribozymes. Potential restrictions of the method and possible ways to overcome them are also pointed out. An in vitro SELEX protocol commonly includes four aspects: initial RNA pool preparation, catalysis processing, in vitro selection and re-construct the initial RNA pool by amplification (Figure 1-1).

Figure 1 - 1 .

 11 Figure 1-1. The common procedure scheme for the in vitro selection of catalytic RNAs.

Figure 1 - 2 .

 12 Figure 1-2. The 3' and 5'-ends of in vitro selected ribozymes from Szostak (A), Suga (B) and Yarus (C) teams. The red square circle indicates where esterification (A, B) and aminoacylation (C) occurs. All the 5'-ends are base-paired and their sequences are part of in the constant region in the initial pools. The 3'-ends are mostly free. "[" indicates the abbreviated parts. A, B and C are adopted from[START_REF] Illangasekare | Aminoacyl-RNA synthesis catalyzed by an RNA[END_REF][START_REF] Lohse | Ribozyme-catalysed amino-acid transfer reactions[END_REF][START_REF] Lee | Ribozyme-catalyzed tRNA aminoacylation[END_REF].

Figure 1 - 3 .

 13 Figure 1-3. Small ribozymes derived from in vitro selection candidates. A: Yarus 29nt ribozyme derived from "Isolate 29 RNA" (Illangasekare and Yarus 1997). B: C3 ribozyme selected by Yarus group in 2009 with three conserved bases (Chumachenko et al. 2009). C: further modified a mini ribozyme derived from C3 ribozyme (Turk et al. 2011).

  -4, A). Ribozyme-catalyzed aminoacylation is also based on this mechanism. The Yarus group has proposed a possible catalytic mechanism for the C3 ribozyme based on computed energy minimization with solvent (Figure1-4, B), where the G14 nucleotide plays the role of the M-B reagent (Figure1-4, A) to catalyze the U26 attack on Phe-AMP and the release of AMP.

Figure 1 -

 1 Figure 1-4. A: Acid-base mechanism of RNA aminoacylation. RL represents the leaving group. "M" Lewis acids. "B:" organic/inorganic bases or electron donors. B: C3 ribozyme and proposed catalysis mechanism.

Figure 1

 1 Figure 1-5. A: Mfold predicted secondary structure of KK13. B: Experimental design of two cooperating ribozymes that process amino acid activation and aminoacylation (Xu et al. 2014).

(

  Figure1-6, A). In modern cells, the aaRS catalyze amino acid activation with ATP through the use of magnesium and arginine or lysine residues to manage the negative charge of the triphosphate (Figure1-6, B). It means even if the aaRS work at apparent pH around 7, there is an electron-deficient environment inside the catalytic center (or Lewis acidic environment).Likewise, Yarus KK13 ribozyme works at mildly acidic pH (i.e. in an H + -rich environment) with 5 mM calcium. Possibly, a calcium-phosphate interaction can neutralize the negative charge of βand γ-phosphate. Moreover, 80% of the α-phosphate group is protonated at pH 4 (Corfù and Sigel 1991), which further decreases the electronic repulsion between acetate and triphosphate groups (Figure1-6, C).

Figure 1 - 6 .

 16 Figure 1-6. Amino acid activation by ATP. A: At physiological pH, both ATP and an aminoacyl group are negatively charged molecules. Nucleophilic attack is impossible. B: aaRS enzymes employ the magnesium as well as positively charged amino acids side chains to bend and significantly reduce the negative charge density of triphosphate to facilitate activation. C: Yarus KK13 ribozyme requires calcium coordination pH=4.0 for optimal activity. Under these conditions, the α-phosphate has been protonated[START_REF] Corfù N A | Acid-base properties of nucleosides and nucleotides as a function of concentration. Comparison of the proton affinity of the nucleic base residues in the monomeric and self-associated, oligomeric 5'-triphosphates of inosine (ITP), guanosine (GTP), and ade[END_REF]. It is possible for aminoacyl group attacking. "(" indicates the repulsive potential surface. Blue color represents a partial enzymatic environment.

Figure 2 -

 2 Figure 2-1 outlines the individual steps constituting these two parts:

Figure 2 - 1 .

 21 Figure 2-1. SELEX procedure: 1) Chemically synthesized DNA templates or treated PCR products which include very short conserved bases are used to transcribed into a pool of random RNA; 2) Pool of RNA incubates with complex buffer; 3) After incubation, RNA is treated by periodate. The 3'-end non-aminoacylated RNA is oxidized. Followed by deacylation, the amino acid is released from the 3'-end of aminoacyl RNA and make the 3'-end available for adapter ligation; 4) 3'-end nonoxidized RNA ligates with a carefully designed RNA adapter carrying a restriction enzyme recognition site by T4 RNA ligase; 5-7) Reverse transcribed DNA undergoes T4 DNA ligation with a dsDNA T7 promoter anchor to construct the full length of cDNA and ready for PCR amplification; 8) Restriction enzyme treatment removes the adapter part and generates the new DNA templates for next round selection.

  bases at the 3'-end RNA Sequence (5'→3') GGN NNN NNN NNN ……… NNN NNN NNN S a CA GGN NNN NNN NNN ……… NNN NNN NNY b CCA a S=C or G; b Y=C or U; Right figure. Template selectivity achieve by the overhang or reverse primers: 1. When template with the correct size is ligated to the adapter, reverse transcription and/or PCR can occur. 2 and 3: when aborted or extended transcripts are ligated to the adapter, mismatch(es) with the overhang of a reverse primer may stop either reverse transcription or PCR. Blue represent selected RNA oligo; Dark purple indicates the conserved bases at the 3'-end; Dark green represents the RNA adapter; The upper strand is the reverse primer (light green = primer; purple = overhang); Red arrow shows the direction of reverse transcription and the red cross symbolizes RT or PCR interruption.

Table 2- 3 .

 3 Templates carrying more constant positions at the 5'-end RNA Sequence (5'→3') GGM a ANN NNN NNN ……… NNN NNN NNY CCA GGC ACN NNN NNN ……… NNN NNN NNY b CCA a M=A or C; b Y=C or U; Right figure. DNA ligation step: 1. ExpectedcDNA (3'→5') ligation with the DNA anchor. 2. Unspecific ligation between the DNA anchor and primer of the adapter. Orange symbol represents the double-stranded DNA anchor with a sticky end of overhang compatible with 3'-end of cDNA, the overhang could be either "GGMA" or "GGCAC" (5'→3') for instance; Light blue is the cDNA sequence transcribed from selected RNA target; Light purple and light green is the primer of the adapter (3'→5'), in this case the purple part represents the sequence "RGGT".

Figure 2 - 3 .

 23 Figure2-3. RNA ligation between the RNA template and RNA adapter. RNA template which is ending by "UCA" (left) shows higher efficiency than "UCG".

Figure 2 - 4 .

 24 Figure 2-4. Modes of metal ion binding to RNA. Cations (Mn + ) can interact with RNA (A, B) requiring at partial and total dehydration of the metal ion, or transient, longdistance interactions between the solvated RNA and metal ion (C) (Johnson-Buck et al. 2011).

  nitrogen and sulfur atoms (Figure2-4). The involvement of magnesium in almost all biochemical processes related to translation is now well established[START_REF] Lightfoot | Magnesium-dependence of in vitro translation programmed by gene-specific mRNAs[END_REF][START_REF] Lancaster | Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site[END_REF][START_REF] Sun | Pentamidine binds to tRNA through non-specific hydrophobic interactions and inhibits aminoacylation and translation[END_REF]: From the tRNA folding and tRNA aminoacylation to ribosomal structure and activity, magnesium directly or indirectly interact with substrates to promote catalysis[START_REF] Al-Karadaghi | The structure of elongation factor G in complex with GDP: conformational flexibility and nucleotide exchange[END_REF][START_REF] Banik | Aminoacylation reaction in the histidyl-tRNA synthetase: fidelity mechanism of the activation step[END_REF][START_REF] Bhaskaran | Kinetics of tRNA folding monitored by aminoacylation[END_REF][START_REF] Petrov | RNA-magnesium-protein interactions in large ribosomal subunit[END_REF]). In relation with the ATP activation issue, the catalytic site of Lysyl-tRNA class II synthetase (Figure2-5) requires three Mg 2+ to present in the amino acid adenylation step by bending the triphosphate groups of ATP into U-shape to expose the α-phosphate for nucleophilic attack[START_REF] Desogus | Active site of lysyl-tRNA synthetase: structural studies of the adenylation reaction[END_REF]. On the other hand, a class I aminoacyl-tRNA synthetase such as Tryptophanyl-tRNA synthetase requires only one Mg 2+ for amino acid activation[START_REF] Retailleau | Crystal Structure of Tryptophanyl-tRNA Synthetase Complexed with Adenosine-5′ Tetraphosphate: Evidence for Distributed Use of Catalytic Binding Energy in Amino Acid Activation by Class I Aminoacyl-tRNA Synthetases[END_REF]. The presence of Mg 2+ ion located near the β and γ phosphate linkage can significantly reduce the negative charge density of the oxygen atoms and thereby aid the nucleophilic attack by the carboxylic group of the amino acid. Resent molecular dynamics simulations research also shows that Mg 2+ can support ribozyme structural stability and possibly facilitating catalysis[START_REF] Ucisik | Molecular Dynamics Study of Twister Ribozyme: Role of Mg2+ Ions and Hydrogen-Bonding Network in Active Site[END_REF]. In our selection experiment, 5 mM MgCl2 are included in the buffer to facilitate potential aminoacylation.

Figure 2 - 5 .

 25 Figure 2-5. Schematic representation of the active site of LysU aaRS (E. coli), showing predicted hydrogen bonds (A) in the ternary complex with lysine and ATP and (B) in the complex with the lysyl-adenylate intermediate. (Desogus et al. 2000).

  (1) RNA + aa-AMP ↔ aa-RNA + AMP………………………. (2) Due to the hydroxyl group being a rather poor active group, almost all successful SELEX investigations on self-aminoacylating ribozymes so far used pre-activated amino acid to bypass the difficult step of activation. Commonly employed leaving groups are AMP, CoA (Coenzyme A), CME (Cyanomethyl ester), CBT (p-chlorobenzyl thioester), DBE (3,5-dinitrobenzyl ester).

  thioesters, acetyl CoA and acyl phosphates. All the aminoacyl tRNA synthetases (aaRS) require ATP as activating cofactor for in vivo aminoacylation of the tRNA. Investigations of aaRS crystal structures have provided many insights into the mechanisms of enzymatic amino acid activation[START_REF] Giegé | Aminoacyl-tRNA Synthetases in the Bacterial World[END_REF]. Usually one to three Mg 2+ ions cooperate with amino acids side-chains in the catalytic site bend the triphosphate of ATP to expose the α-phosphate to the acyl group of the amino acid substrate. Concurrently, ions and other positively charged functional groups act as strong Lewis acid to reduce the electron density of the triphosphate in order to facilitate the nucleophilic attack by the acyl group (Figure2-6).

Figure 2 - 6 .

 26 Figure 2-6. Schematic showing three stages of hydrogen bonding interaction of the active site residues with the reacting substrates of His-aaRS from E. coli. Reactants (left), intermediate (middle), and activated amino acid (right) (Banik and Nandi 2010).

  Figure 2-7. A: Stereochemistry of peptide-RNA construction. The peptide (inside)binds the RNA molecule (outside) into double helixes and stabilized by the hydrogen bonds between peptide carbonyl and the ribose 2'-OH groups, between amide nitrogen and water molecules (blue spheres) between the ribose O1 and 2'-OH groups[START_REF] Carter | What RNA World? Why a Peptide/RNA Partnership Merits Renewed Experimental Attention[END_REF]. B: poly-L-lysine, poly-L-arginine and spermine.

Figure 2 - 8 .

 28 Figure 2-8. Schematic diagram shows showing the result of an OD procedure.

Figure 2 - 9

 29 Figure 2-9 The mechanism of sodium periodate oxidation and β-elimination.

  selection of aminoacylated RNA was established with so-called Oxidation-Deacylation and Deacylation-Oxidation (OD-DO) assays. These assays allow us to select aminoacylated RNA (OD) and also give a negative control (DO) to the procedure. Two additional positive controls (D and NT, no treatment) were also implemented. A similar protocol known as OXOPAP assay has been formerly used by Dittmar et al. and Gaston et al. to quantify aminoacylated tRNA (Gaston et al. 2008; Puerto-Galá n and Vioque 2012; Dittmar et al. 2005). We use the same strategy with optimized condition to select our targeted ribozymes. RNA pools that underwent an aminoacylation assay (Section 2.3.5) are split into different batches for different oxidation and/or deacylation treatment (Figure 2-10) 1) In one batch of experiments, we first treat the RNA sample with NaIO4. Oxidation followed by deacylation (OD) will allow us to select aminoacylated RNA, and thus any potential ribozyme.

Figure 2 - 10 .

 210 Figure 2-10. Outline of OD-DO assay. (1), (2), (3), (4) stand the experiment workflow of OD, DO, D, NT, respectively.

Figure 2 - 11 .

 211 Figure 2-11. Gel analysis of total RNA. Left gel shows the total RNA loaded on agarose gel after OD-DO treatment. The lane "DO" shows an obvious disappearing of RNA. Right gel shows the same total RNA loaded on agarose gel after DNase treatment. The lane "DO" is showing up again.

Figure 2 -

 2 12 shows an oxidation experiment results.

Figure 2 - 12 .

 212 Figure 2-12. The final RT-PCR result of mini-tRNA Ala (1, 2), Val (3, 4) and Gly (5, 6) after treat with NaIO4. Clearly, after oxidation the lane 1, 3, 5 should have shown a negative result of RNA ligation. Lane 1, 3, 5 represent the PCR products which generate from the ligation of mini-tRNA and RNA adapter. Lane 2, 4, 6 represent the PCR product of mini-tRNA themselves (Ala, Val and Gly) as a control. Red arrow points the primer-dimer by-product.

Figure 2 -

 2 Figure 2-13. Left: the commonly used protecting group during chemically RNA synthesis. Right: lane 7 shows the NaIO4 treated RNA has much lower ligation efficiency than the non-treated one (lane 9). Lane 8 shows a negative control which didn't do the RNA ligation. Red arrow points the primer-dimer by-products.

coli

  BL21 by acid phenol method (Supporting information). Acidic pH prevents deacylation during extraction. An amount of 4 µg total RNA was loaded into 4 tubes and went through identical DO/OD/D/NT treatments. To prevent genome amplification, all the samples were subsequently treated with DNase I at alkaline pH. The final 20 cycles PCR shows that a similar difference if observed between OD and DO experiments.

Figure 2 -

 2 Figure 2-14. tRNA Tyr from E. coli is employed as positive control to verify the protocol. tRNA Tyr is a specific 85 nt long natural tRNA. After OD-DO assay treatment, it will subsequently ligate with a carefully designed RNA adapter P with 23 nt in length. By using tRNA Tyr 5'-reverse primer and primer of the adapter, the expect products should show a size at 108 bp. Left: Aminoacyl tRNA Tyr yield by in vitro aminoacylation with commercial purified natural E. coli tRNA Tyr , Tyrosine, and E. coli Tyrosine-specific aaRS. Gel shows the 20 cycles PCR product of in vitro selected aminoacyl tRNA Tyr . 4 lanes represent OD/DO/D/NT treatment. Right: 1 µg total RNA extracted from E. coli at OD600 0.4 is treated by OD/DO/D/NT procedure. Gel shows the final 20 cycles PCR result.

a

  All the tubes covering with aluminum films for oxidation b Dissolve 5.4 mg NaIO4 in 250 μl water to prepare 0.1 M sodium-periodate solution c After oxidation, add 10 μl Glucose (0.5 M) and incubate 15 min on ice. Then add 1 μl Glycogen as well as 6 μl DTT (0.1 M) into the oxidation solution.

  RNA adapters were successively tested to optimize the ligation reaction and the subsequent RT-PCR protocols. They can be categorized into 4 classes based on their sequence and/or structure profiles (Figure 2-15). In the first class, all adapters are single stranded. This class includes adapter J, K, L, XL, and M (former tests with adapter A to I were performed by Maser student Lauriane Cacheux). All other adapters are RNA-DNA hybrids. Among those RNA-DNA hybrid adapters, class II and class IV are similar except for the length of the single stranded portion of the RNA strand and the restriction site, while class III adapter includes a partially noncomplemented DNA strand. These chimeric adapter molecules (class II to IV) were designed to prevent unspecific priming on the random track during reverse transcription (See below).

Figure 2 -

 2 Figure 2-15. 1: 5' phosphate adapters tested during the establishment of the protocol. Class I adapters are all single stranded RNA adapter, including adapter J, K, L, XL, and M; 2: Class II adapter is an RNA/DNA hybrid, that includes adapter Hyb-XL; 3: Class III adapters have similar structure to class 2 except the hybrid DNA part has an overlap with the constant 3'-end of the targeted RNA, such as adapter N and NR; 4: Class IV adapters are similar to class 2 but carry a different restriction site. It includes adapter O and P. Sequence details seeTable 2-8.

Figure 2 - 16 .

 216 Figure 2-16. The three colors line represent cDNA (3'→5'): Light green is the primer of the adapter part initially come from the ligated RNA adapter; Light orange is the cT7 promoter sequence which is added by DNA ligation; Light blue is complementary DNA of the random RNA. Dark green is the primer of the adapter forward primer. Brown is T7 promoter reverse primer (5'→3').

Figure 2 - 17 :

 217 Figure 2-17: Random priming during reverse transcription. Light blue lines represent primers of the adapter (3'→5'); The dark green line is the ligated RNA adapter (5'→3'); The dark blue line stands for the random RNA pool (5'→3'); The red arrow indicates the reverse transcription orientation.

Figure 2 - 18 .

 218 Figure 2-18. Correct folded adapter (Left) and adapter duplex (Right). The green part represents the RNA and the light blue part shows the complementary DNA primer as well as a poly-dT loop.

Figure 2 - 19 .

 219 Figure 2-19. Bioanalyzer traces of annealed Adapter Hyb-XL in different concentrations (µM). A) 0.1; B) 1; C) 5; D) 10. Adapter Hyb-XL diluted in 100 µl water was incubated at a pre-heated PCR machine at 90°C for about 2 min and immediately dropped into liquid nitrogen. A sample volume of 0.1 µl was loaded on bioanalyzer (small RNA6000 chip).

Figure 2 - 20 .

 220 Figure 2-20. Bioanalyzer traces of RNA ligation between Adapter Hyb-XL and random RNA N37 before ligation (left) and after ligation (right). Left: 1 µM pre-annealed adapter mixing with random RNA N37 at a final molar concentration ratio 10:1. Right: Given such mixture, bioanalyzer shows the signal after RNA ligation at 4°C overnight. The red arrow represents the pre-annealed adapter Hyb-XL; The dark blue arrow shows the random RNA N37 pool; The green arrow indicates the expected ligation products.

Figure 2 - 1 ,

 21 step 6

Figure 2 - 21 .

 221 Figure 2-21. The main by-product ligation mechanism: the RNA tail of this hybrid adapter degrades to the "rGrG" part (red dot) and reverse transcribed cDNA carrying a 3'-"CC" ending (dark red) can ligate with the DNA T7 promoter anchor (orange). Dark blue represents RNA target, light blue is the DNA part of adapter, green shows the RNA part of adapter, red arrow indicates the reverse transcription orientation.

Figure 2 - 22 .

 222 Figure 2-22. Adapter with 3' DNA overhang: unspecific priming during reverse transcription. Light blue: primer of the adapter; Green: RNA adapter; Dark blue: ligated RNA; Red arrow: reverse transcription orientation.

  high stability of Adapter P folding is made possible by rGrArGrCrCrG/CTCGGC base pairing near the 5'-end of the adapter (Figure 2-25). Instead of poly-U, an adenosine-rich 5'-end (rArGrArA), including some bases of the EarI restriction site is used for RNA ligation. It turns out that EarI could efficiently digest our PCR products, and provide new templates of sufficient quality. And finally, the EarI digestion can achieve to a good quality (Figure 2-26).

Figure 2 -

 2 Figure 2-26. Gel analysis of before (left) and after (right) EarI digestion of the PCR product after one round of selection. 1 µg PCR product (4 libraries) was treated by EarI FastDigest kit (NEB) overnight. Red arrows indicate the digest products.

  -1). The design made it necessary to ligate a second adapter after reverse transcription to allow a final PCR. In any case, a T7 promoter must be added to allow the next round of in vitro transcription. We have tested several DNA duplexes (we call it DNA anchor) with the aim to achieve DNA ligation with the 3'-end of the cDNA and also prevent PCR side products, especially primer dimers. These DNA anchors can be roughly categorized into 4 classes (Figure2-27). The initial design (class I) is a double stranded T7 promoter oligo containing a sticky 3'-"GG" overhang intended to capture the 3'-"CC" end of the selected cDNA target. Classes II and III are identical to class I except for an extended sticky end region composed of a few additional constant positions following the GG overhang and either random bases (Class II) or a poly-Inosine (Class III). The role of these extensions is to help bind the selected cDNA and thus allow a better yield of ligation.We also tried a single stranded version of Class III (Class IV).

Figure 2 -

 2 Figure 2-27. Four classes of DNA ligation anchors. These constructs are used to allow the ligation of the cDNA to a 5' adapter (T7 promoter) with a DNA ligase. Class I is a classic double stranded T7 promoter sequence with a "GG" sticky end. Class II is similar with class I but has a stretch of complementary and random bases following the "GG" sticky end. Class III has inosines instead of random bases. Class IV is a single stranded DNA oligo constituted by T7 promoter sequence, a cDNA overhang sequence and a poly-inosine tail. The T7 promoter sequence is in dark blue. The red dot stands for the 5'-end monophosphate group; The orange part indicates a sequence complementary to the targeted cDNA 3'-end.

  T7 promoter, we tried to implement the addition of Class IV anchor during PCR with a specific annealing step. This procedure was taking advantage of the fact that a Taq DNA polymerase (ThermoFisher) still has some activity at low temperature (Figure2-28).

Figure 2 -

 2 Figure 2-29. T7 promoter adding by Taq DNA polymerase: the mechanism of annealing-elongation procedure. Green: reverse transcribed target cDNA (3'→5'); Dark blue: T7 promoter (5'→3'); Dark blue dash line: elongated cT7 promoter sequence; Red line: poly-inosine tail (5'→3').

Figure 2 -

 2 Figure 2-30. Primer-dimer generated by reverse transcription. Reverse transcriptase, like M-MLV RT, has a non-templated nucleotide addition activity. It preferably adds G or C at the cDNA's 3'-end. 3' Adaptors with added cytidines become compatible with the DNA anchor and undergo DNA ligation to generate a primer dimer. The green line represents the DNA oligo; The red line represents RNA oligo; Blue balls indicate the monophosphate groups; Red stop symbolizes an oligo modification preventing ligation.

Figure 2 -

 2 Figure 2-31. The Mung bean blunt enzyme only digests the single stranded RNA and DNA. Red color represents the RNA oligos; Green color indicates the DNA oligos; Dash line shows the digested single stranded RNA or DNA.

Figure 2 -

 2 Figure 2-32. Random RNA pool N37 (37 nt) ligated with adapter P and transcribed into cDNA with SSIII RT. A purified cDNA sample was divided into two groups, with (3 and 4) or without (1 and 2) Mung bean treatment before DNA ligation. Lanes 1 and 3 show the PCR products obtained with T7 promoter primer (25 nt) & primer of the adapter (27 nt), while lanes 2 and 4 show the result of PCR using T7 promoter+GGCACGA overhang (30 nt) & primer of the adapter+TAG overhang (30 nt). Only the sample (lane 4) treated by blunt enzyme and amplified by overhanged primers can generate the expected PCR product without any side-product.

Figure 2 -

 2 Figure 2-33. cDNA structure and T7 promoter overhang primer design. The upper strand briefly shows the structure of the cDNA. The green part represents the added adapter part; The purple and orange parts indicate the conserved sequence of the initial RNA template; The light yellow part shows the random track of the initial RNA template. The bottom strand shows the structure of the T7 promoter with overhang primer. The blue part represents the T7 promoter sequence and the orange part indicates the overhang sequence priming on the upper cDNA orange part.

Figure 2 -

 2 Figure 2-33. Obviously, only 7 base pairs are not sufficient enough to provide strong binding during the annealing step. However, the predicted melting temperature is around 25ºC in a standard PCR mixture with 2 mM MgCl2, implying that we could take advantage of the low activity of Taq DNA polymerase at room temperature (see Section 2.6). This design was initially tested with a ssDNA similar to the considered cDNA. T7 promoters with three different overhang (7, 8 and 9 nt) were tested using both a Taq DNA polymerase (ThermoFisher) and a Taq Hot Start DNA polymerase (NEB). When the annealing temperature was set at 60ºC, only the T7 promoter with a 9-nt overhang could produce efficient amplification with the Hot Start Taq polymerase (Figure2-34, 3'), while the 7 nt overhang T7 promoter only generate a very faint signal. When the PCR mixture is prepared at room temperature with a Taq DNA polymerase, the final PCR can yield strong and specific signal with 7-, 8-, and 9-nt overhang, indicating that the considered (GC-rich) 7 nt overhang is long enough to prime and PCR the cDNA. Remarkably, no unspecific product is generated (Figure2-34). This experiment demonstrated that RNA pools with as little as 7 constant positions at the 5' end and 3 constant positions at the 3' could undergo specific PCR amplification after selection.

Figure 2 - 34 .

 234 Figure 2-34. Short primers PCR procedure test. Left: PCR products with DreamTaq DNA polymerase (ThermoFisher) using T7 primers with 7 nt (1), 8 nt (2) and 9 nt (3) overhangs together with a forward primer. Right: PCR product with OneStart Taq DNA polymerase (NEB) using T7 primers with 7 nt (1'), 8 nt (2') and 9 nt (3') overhangs together with a forward primer.

Figure 2 -

 2 Figure 2-35.The final SELEX protocol. 1) Chemically synthesized DNA templates or treated PCR products which include very short conserved bases are used to transcribed into a pool of random RNA; 2) Pool of RNA incubates with complex buffer; 3) After incubation, RNA is treated by periodate. The 3'-end non-aminoacylated RNA is oxidized. Followed by deacylation, the amino acid is released from the 3'-end of aminoacyl RNA and make the 3'-end available for adapter ligation; 4) 3'-end nonoxidized RNA ligates with a carefully designed RNA adapter carrying a restriction enzyme recognition site by T4 RNA ligase; 5) Ligated RNA is reverse transcribed to cDNA; 6) 7nt overhang short primer PCR to add the T7 promoter sequence; 7) Restriction enzyme treatment removes the adapter part and generates the new DNA templates for next round selection.

  5) went through 4 to 7 cycles of selection (Figure 2-36), during which they were incubated with a mixture of 19 different kinds of free amino acids with ATP and GTP as activating cofactors. In order to quickly proceed through the cycles, only the OD treatment was achieved during the selection step; the DO, D and NT controls (Section 2.4.2) were done only at the end, after a convergence was detected. The initial selection started with a pool of RNA including approximate 10 13 RNA molecules in each library. (Table

  reaction, all four samples underwent the same OD treatments and were amplified by RT-PCR. PCR amplification after the 1 st cycle of selection was not easy to achieve due to the very low amount of remaining (non-oxidized) RNA after OD treatment. The usual amount of solution from the reverse transcription reaction (2 µl out of 20 µl in total) had to undergo as many as 35 cycles of PCR to only generate a faint signal on gel. In total, 5 tubes of 50 µl-PCR reaction were combined for gel purification.The quality and diversity of the pools was verified after the first round selection. The PCR products were used in a cloning experiment using the CloneJET kit (ThermoFisher), and about 10 individual clones in each pool were sent for sequencing. The sequencing results for each pool are shown in Table3-2, A-D (the red letters highlight the constant regions). As expected at this stage, the 35 sequences in total are all different and do not show any similarity either in sequence or in their predicted secondary structure (established using Mfold; Zuker 2003).

  ................ GGCACGACUACCACACUCA ................... GGCACGAUGUACCUUUGUCA ((((...........)))).

  ....))))........... GGCACGAGCCGGCAACUCA (((....)))......... GGCACGAACAACACAGUCA ...................Table 3-2, B. Sequencing results of N23 pool after 1 st cycle selection Sequence Optimal secondary structure GGCACGACCACAUUACGGCCUCA(((.((.........)))))... GGCACGAGCCCACCGCGAGAUCA (((....)))............. GGCACGAAACGACUCUGCCUUCA ((((.((......)))))).... GGCACGAAUUACAUUUAGACUUUUUCA ........................... GGCACGACACAAUGCCGUCCUCA ((((........))))....... GGCACGAUUCUACCAAUAGUUCA ....................... Table 3-2, C. Sequencing results of N30 pool after 1 st cycle selection Sequence Optimal secondary structure GGCACGAAGGAGCUUGCGUAGUGUCA (((((...(.......)...))))). GGCACGAUCAUCCCCUCUUGAAUAUCA .......(((........)))...... GGCACGAAUCCUAACCCAAGUCCCCCUUCA ((.((..............)).))...... GGCACGAGUCGAUCUUUCCGCCAAUUGUCA (((..(((.....)))...)))........ GGCACGACAUGGCCCUCCCCAGAAUAUUCA (((........)))................ GGCACGAUGCCGCGGGUUCGGGCGCUAUCA ((((...))))(((........)))..... GGCACGAACAAACAUGUUACGCGCUCCUCA (((.((((((....)))).)).)))..... GGCACGAGGGCACGAACAAGUUAUUGCUCA .......(((((............))))).

  GGCACGACCGUAUUGUGAGGCCGUGUGUGAUCA .(((((.((.........)).)))))....... Table 3-2, D. Sequencing results of N37 pool after 1 st cycle selection Sequence Optimal secondary structure GGCACGAUAUCGUCGCCUCCUCA((((((....))).)))...... GGCACGAACCCAACGCGGUCUCGCCCUCA (((..((.((......))))..))).... GGCACGAUGUUGAAGUACUAGUACAUCCCGCCUCA (((..(((((............)))))..)))... GGCACGAACACACAAUUCACCGGACUUCCAAGUGUCA (((((..........................))))).

  GGCACGACCCAUUGCACAUUAACGCUCACCCUAGUCA .(((........)))......................GGCACGACAACUACUCGCCCCGUGAGUCUACAUGUCA.....((((...((((((...)))))).....)))).

  -1, left). The PCR products were cleaned up and digested with the EarI restriction endonuclease (NEB) (Figure 3-1, right).

Figure 3 - 1 .

 31 Figure 3-1. Left: After the first selection cycle, 1 µg of PCR products were loaded on 4% agarose gel. Right: The same PCR products (1 µg) after digestion by the EarI restriction enzyme at 37ºC overnight.

Figure 3 - 2 .

 32 Figure 3-2. Bioanalyzer signals of initial RNA pool (left) and RNA pool after the first selection cycle (Right). A longer transcript is commonly observed from the second round.

Figure 3 - 3 .

 33 Figure 3-3. Bioanalyzer traces of the initial pools and the final pools. The N23 library went through 4 cycles of selection; the N19 and N30 library were run until they reached the 6 th selection cycle; the N37 pool was further pushed to the 7 th cycle. Note that the scale of the x-axis is not uniform.

  .......))).......... 21 GGCACGACGUACGAUCCAUGUCA 1 .....(((((.......))))).

Figure 3 - 4 .

 34 Figure 3-4. Predicted secondary structure (Mfold) of the most enriched sequences in N19 library. A: the "Key" shape secondary structure of No.2 (10 copies); B: the "Hammer Throw" shape secondary structure of No.17 (12 copies).

  ......)))............

  ......)))...........11GGCACGACUCUGCGAGGCCCUCA 1 (((.((......))..))).... 12 26nt GGCACGACUAGUGCAGAGCAACCUCA 1.((((.....)))).(((....))).

  .....))).))....(((......))).

  .......))).......... GGCACGACUAGACAGCCAACUCA 11 (((...........)))...... GGCACGAGACACCUACCGCCUCA 7 (((...((....))...)))... GGCACGAGACAUCUAUCGCCUCA 1 (((...((....))...)))...

  ........)))................ 34nt GGCACGACCAGCGUUCCUCUGGACCCCUACGUCA 12 ((.(((.....))).))....(((......))).

36nt GGCACGACCAGCGUUCCUCUGGACCCCUACGUCUCA 6 (

 6 (.(((.....))).))...((((......))))..37ntGGCACGACCAGCGUUCCUCUGGACCCCUACGUCUUCA 7((.(((.....))).))...((((......))))...

  ): most sequences share some similarities in their secondary structures (e.g. Table3-8, No.10 to 13), such as the one shown in Figure 3-5.

Figure 3 - 5 .

 35 Figure 3-5. Optimal secondary structure (Mfold) of one of the enriched sequence No.10 (7 copies) inTable 3-8.

Figure 3 - 6 .

 36 Figure 3-6. Optimal secondary structure of No. 21, 22 and 23 in Table 3-8. Their 5'end hairpins are the same while point mutations present at the 3'-end can slightly change the size of stem as well as the length of unstructured 3'-end residues.

Figure 3 - 7 .

 37 Figure 3-7. Left: Length distribution of libraries N19, N30 and N37. Right: Normalized length distribution for all three libraries. The X axis stands the length of sequences. The Y axis is the number of sequences.

  ...........)))....N19(6r)GGCACGACUAGACAGCCAACA 3 (((...........)))....N19(6r)3.3 Tests of self-aminoacylation with the selected librariesBefore we knew about the "mini-monster" phenomenon, we decided to verify the aminoacylation activity of the pools of RNA and also test some specific sequences. We first tested library N19. Two parallel experiments were setup, in which the amino acids mix was present only in one of them (Figure3-10). It was expected that the one with the amino acids should show a positive selection with the OD treatment while the experiment without amino acid should provide a negative signal in the absence of aminoacylation. Figure 3-10 shows that both the OD and DO treatments gave a negative signal regardless of the presence of amino acids.

Figure 3 - 10 .

 310 Figure 3-10. Test for aminoacylation with N19 library after 6 rounds selection.

Figure 3 -

 3 Figure 3-11. HPLC traces of libraries N23, N30 and N37 after incubation.

Figure 3 - 12 .

 312 Figure 3-12. Verification of aminoacylation for the library N37 after 7 rounds selection.

Figure 3 -

 3 13 shows two examples of the HPLC analysis.

Figure 3 -

 3 Figure 3-13. HPLC analysis for specific sequence aminoacylation test.

  approach relies on total RNA extraction under non-deacylation conditions (essentially while keeping a low pH during the procedures). After extraction, RNA samples follow different series of treatments: OD (oxidation-deacylation), DO (deacylation-oxidation), D (deacylation only) and NT (no treatment) as is described in last chapter. Treated samples are processed to generate cDNA banks through the addition of adaptors, and these banks are sent to a company for deep sequencing using the Illumina pair-end sequencing technology. A comparison between batches is expected to provide the information to identify aminoacylated species.In contemporary cells, tRNA is the prototypical molecule of aminoacylated RNA. Its primary function is to participate in translation on the ribosome, where proteins are being built according to the rules of the genetic code. More than 20 different variants of this molecule are specifically aminoacylated by aminoacyl-tRNA Synthetase[START_REF] Giegé | Aminoacyl-tRNA Synthetases in the Bacterial World[END_REF]. In bacteria, aminoacylated tRNA not only participate in translation; they are also involved in cell wall metabolism[START_REF] Raina | TRNAs as regulators of biological processes[END_REF]. Furthermore, prokaryotic cell uses several strategies to maintain appropriate levels of aminoacylation through the use of different sensors such as the T-box riboswitch (mainly in Firmicutes). The study of the level of aminoacylation with an OD-DO methodology could thus highlight new information about this essential actor in cell metabolism. Furthermore, because tRNA molecules constitute a large fraction (15%) of total RNA in bacteria(Uzman 2001), tRNA is an obvious candidate to validate and calibrate our protocol.

(

  Imagif). A home-made pre-purification protocol by gel electrophoresis, established in collaboration with the technician at the Facility (Erwin Vandijk), was implemented to obtain three samples belonging to different size categories: 10-50 nucleotides, 50-100 nucleotides and 100-250 nucleotides. It is in order to normalize the weight of each category, and prevent the 50-100 nt category (containing most of the tRNA) to become overwhelmingly dominant. For each category, an RNA spike of appropriate size was designed, and a normalized mixture of the three spikes was added to the processed samples before gel purification. Gel purification of the OD, DO, D and NT samples resulted in 12 different samples, each of them being labelled with an index sequence corresponding to the Illumina sequencing requirement during bank preparation. Bank preparation was achieved by the consecutive ligation of universal adaptors (with T4 RNA ligase) to both the 5'-end and the 3'-end (Figure 2.1). A polynucleotide kinase (PNK)

Figure 2 . 1 :

 21 Figure 2.1: Illumina TruSeq Small RNA library standard preparation (work flow). Dark blue: 3' RNA adapter and lighter color represents the complementary DNA sequence; Dark green: 5' RNA adapter and lighter color indicates the complementary DNA sequence; Purple: target RNA fragments.

Figure 2 . 2 .

 22 Figure 2.2. Bioanalyzer traces of treated RNA sample: 1-4 number represents OD, DO, D and NT respectively. A diluted sample from the recovered samples were injected for analysis.

Figure 2 . 3

 23 illustrates the results with a window analysis encompassing a methionine tRNA operon (MetZ, MetW and MetV). While taking into account the spike correcting factor, all three tRNAs are covered by approximately the same number of reads regardless of the OD, DO, D or NT treatment (Table 2.5).

Figure 2 . 3

 23 Figure2.3 shows why OD and DO treatments fail to remove tRNA reads from the corresponding banks: although RNA fragmentation was purposely not implemented after the treatments, the large majority of the reads (regardless of the RNA identity) are fragments of full-length RNA molecules, revealing that fragmentation still significantly occurred during bank preparation, i.e. after oxidation and deacylation treatments. Fragmentation may generate free 3'-ends available for adaptor ligation; these fragments therefore may be included in the banks.

Figure 2 .

 2 Figure 2.3. OD, DO, D and NT reads mapping a methionine tRNA operon (MetZ, MetW and MetV genes, in dark blue). Reads (in brick color; alternate colors highlight read mismatches) are classified with the longest ones on top. Only the upper reads (constituting a small fraction of all reads) are visible. Gene coverage in shown on top (in grey).

Figure 2 . 4 .

 24 Figure 2.4. DO and NT reads mapping the valY gene (a valine tRNA, in dark blue). Reads (in brick color; alternate colors highlight read mismatches) are classified with the longest ones on top. Only the upper reads (constituting a small fraction of all reads) are visible in the two DO and NT windows. Spike normalization (DO = 1043 x 0.6614 = 690; NT = 609 x 1.0039 = 611. See Table 2.5) reveals that both treatments have a similar coverage. Gene coverage in shown on top (in grey).

Figure 2 . 5

 25 shows in fact that with OD, D and NT treatments, already half of the reads lack the terminal adenosine. Although we could not explain this feature, it appears that the nature of the termination might be connected with aminoacylation, and may still allow us to quantitatively compare OD and DO samples.

Figure 2 .

 2 Figure 2.5. OD, DO, D and NT reads mapping a MetV gene (methionine tRNA). This magnification of Figure 2.3 reveals that almost all CCA 3' end of DO treated tRNA reads lack the terminal adenosine, while this base is missing in approximately half of the reads with the other treatments.

Figure 2 . 6 .

 26 Figure 2.6. Nature of the 3' termination in E. coli tRNA reads. A) Dispersion of the CCA/(CC+CCA) ratio in all tRNA genes, NT sample (in alphabetical order): [alaT,alaU, alaV, alaW, alaX, argQ, argU, argV, argW, argX, argY, argZ, asnT, asnU, asnV, asnW, aspT, aspU, aspV, cysT, glnU, glnV, glnW, glnX, gltT, gltU, gltV, gltW, glyT, glyU, glyV, glyW, glyX, glyY, hisR, ileT, ileU, ileV, ileX, leuP, leuQ, leuT, leuU, leuV, leuW, leuX, leuZ, lysQ, lysT, lysV, lysW, lysY, lysZ, metT, metU, metV, metW, metY, metZ, pheU, pheV, proK, proL, proM, selC, serT, serU, serV, serW, serX, thrT, thrU, thrV, thrW, trpT, tyrT, tyrU, tyrV, valT, valU, valV, valW, valX, valY, valZ]. Due to low number of read counts (< 40),[IleX, lysQ, lysT, lysV, lysW, lysY, lysZ] values are not shown. The average value (red line) is 0.672. B) Correlation of the CCA/(CC+CCA) ratio between untreated sample (NT) and the OD, DO and D samples. The linear regression of the D sample has a slope of ~1.0.

  Figure 2.6. Nature of the 3' termination in E. coli tRNA reads. A) Dispersion of the CCA/(CC+CCA) ratio in all tRNA genes, NT sample (in alphabetical order): [alaT,alaU, alaV, alaW, alaX, argQ, argU, argV, argW, argX, argY, argZ, asnT, asnU, asnV, asnW, aspT, aspU, aspV, cysT, glnU, glnV, glnW, glnX, gltT, gltU, gltV, gltW, glyT, glyU, glyV, glyW, glyX, glyY, hisR, ileT, ileU, ileV, ileX, leuP, leuQ, leuT, leuU, leuV, leuW, leuX, leuZ, lysQ, lysT, lysV, lysW, lysY, lysZ, metT, metU, metV, metW, metY, metZ, pheU, pheV, proK, proL, proM, selC, serT, serU, serV, serW, serX, thrT, thrU, thrV, thrW, trpT, tyrT, tyrU, tyrV, valT, valU, valV, valW, valX, valY, valZ]. Due to low number of read counts (< 40),[IleX, lysQ, lysT, lysV, lysW, lysY, lysZ] values are not shown. The average value (red line) is 0.672. B) Correlation of the CCA/(CC+CCA) ratio between untreated sample (NT) and the OD, DO and D samples. The linear regression of the D sample has a slope of ~1.0.

Figure 2 . 7 .

 27 Figure 2.7. CCA/(CC+CCA) ratios for OD, DO and D samples normalized by the ratios of the NT condition in all tRNA genes (Figure 2.6). This plots further highlights the neutral effect of the D treatment (the average value of the ratio is ~1). The four circles dots correspond to the OD values of methionine tRNA genes metV, metW, metY and metZ.

  Relative abundance of tRNA transcriptsRelative proportion of [CC + CCA] tRNA reads occur. In the following Section, we propose a new method of small RNA sequencing to specifically evaluate the aminoacylation level of any RNA transcript from total RNA.

Figure 2 . 9 :

 29 Figure 2.9: Removing methylated nucleotides to sequence tRNAs. Whereas reverse transcriptase (RT) cannot extend through a fully modified tRNA (left), treatment with AlkB removes select methylated nucleotides to allow efficient reverse transcription and deep sequencing (center and right). Full-length and truncated cDNAs are shown in purple and green, respectively (Zheng et al. 2015).

  Figure 2.10 shows the work flow of the new protocol. Treated RNA are reverse transcribed into cDNA right after 3' adapter ligation. A gel purification of the DNA/RNA duplex enables us to remove the excess of 3' adapter and select transcripts within a size range of interest (from 35 to 300 nt).

Figure 2 . 10 .

 210 Figure 2.10. Work flow of homemade aminoacyl RNA sequencing library preparation. Dark purple: treated RNA samples; Dark blue: pre-adenylated DNA adapter; Red: poly-A tail; Green and light blue: Illumina TruSeq required oligos.

Figure 2 .

 2 Figure 2.14. Gel analysis of the first PCR amplification with Illumina RT primer with dT17 tail and primer of the adapter.

Figure 2 . 15 .

 215 Figure 2.15. Gel analysis of a final PCR with 20 cycles (pre-test final cDNA bank, NT samples from batch 1 to 3)

Figure 2 .

 2 Figure 2.16. Left: Final PCR products size checking on 4% agarose gel. Right: PCR control with tRNA Tyr .

Figure 2 . 17 .

 217 Figure 2.17. Bioanalyzer trace of a final PCR (final cDNA bank and one example of the NT sample)

Figure 2 . 18 .

 218 Figure 2.18. Single clone sequencing result of "B-2" OD sample. Blue shows the insert sequences. Black letter is the tRNA Leu full sequence and red letter indicates the base modification. #: 2'-O-methylguanosine; T: 5-methyluridine; D: dihydrouridine; P: pseudouridine; K: 1-methylguanosine. (http://modomics.genesilico.pl/)

Figure 2 . 20 .

 220 Figure 2.20. The prepared bank libraries are amplified by 35 cycles PCR via different primers. Left: the standard Illumina TruSeq small RNA library kit primers. Right: modified Illumina TruSeq small RNA library kit RT primer with "TGG" overhang. Red arrows point the position of primer-dimer (73 bp).

Figure 2 . 21 .

 221 Figure2.21. Aminoacylation kinetics of transcripts of tRNA Leu and tRNA Leu mutants from E. coli.[START_REF] Zhou | Role of tRNA amino acidaccepting end in aminoacylation and its quality control[END_REF]

  5 Centrifugal Filter Millipore spin column to clean the reaction solution (50 µl reaction solution + 450 µl water, spin 15 min; refill 450 µl water, spin 15 min, collect leftover). Precipitate overnight and suspend the pellet into 20 µl H2O The second round PCR and Index adding PCR mix: 10X AccuPrime buffer II 5 µl, Universal Primer (10 µM) 1 µl, Illumina Index Tag 1~16 (10 µM) 1 µl, Pre-PCR Template (1~16) 100 ng, AccuPrime Taq polymerase 1 µl, H2O fills up to 50 µl. (94°C 2 min; [94°C 30 s, 60°C 40 s, 68°C 20 s] x 10; 68°C 2 min)Gel purificationUsing 30K Amicon ® Ultra-0.5 Centrifugal Filter Millipore spin column to clean the reaction solution (50 µl reaction solution + 450 µl water, spin 15 min; refill 450 µl water, spin 15 min, collect leftover). Prepare 1.2% agarose gel with ethidium bromide. Load PCR products and run the gel with fresh 0.5X TAE buffer at 135 voltages for 25 min. Cut the gel from 150 bp to 400 bp and break the gel by gel breaker tube. Suspend crushed gel into 450 µl water + 50 µl TE buffer and shake at 37°C overnight. Precipitation and re-suspend sample in pure water and verify the concentration.

Table 1 .

 1 Ribozymes with RNA elongation property

	No. Function	pH	Cofactor	Reference
	1	RNA polymerization	8.0	Mg 2+	(Ekland and Bartel 1996)
	2	Template-directed polymerization	8.0	Mg 2+	(Johnston et al. 2001)
	3	RNA polymerization	8.5	Mg 2+	(Zaher and Unrau 2007)
	Because we aim to isolating auto-aminoacylating ribozymes that must bind ATP and/or

GTP as well as amino acid(s), it is appropriate to examine existing single-stranded DNA or RNA molecule that can specifically bind targets such as ATP, peptide or any other organic compound with high affinity. Such oligonucleotides obtained through in vitro selection are called "Aptamers". Aptamers are obtained with in vitro screening procedures that are similar to those designed for ribozymes. With new high-throughput selection and sequencing methods, aptamer libraries are now increasing exponentially

[START_REF] Dupont | Characterisation of aptamer-target interactions by branched selection and high-throughput sequencing of SELEX pools[END_REF][START_REF] Fraser | Oligonucleotide functionalised microbeads: Indispensable tools for high-throughput aptamer selection[END_REF]

. Even though aptamers are not catalytic RNA, their binding motifs can help us understand some ribozyme catalytic mechanisms. In relation with our work, we are interested in amino acid aptamers and ATP aptamers (Table

2

).

Table 2 .

 2 In vitro selected aptamers targeted by ATP/AMP and amino acids

	No. Target	pH	Cofactor	Reference
	1	ATP	7.6	Mg 2+	(Sassanfar and Szostak 1993)
	2	ATP/CoA	4.0	Mg 2+ , Mn 2+	(Burke and Hoffman 1998)
	3	cAMP	7.5	Mg 2+ , Mn 2+ , Ca 2+	(Koizumi and Breaker 2000)
	4	ATP	7.12	Mg 2+	(Vaish et al. 2003)
	5	ATP	7.4	Mg 2+ , NTPs, Spermidine (Sazani et al. 2004)
	6	ATP	7.6	Mg 2+	(Huang and Szostak 2003)
	7	GTP	7.3	Mg 2+	(Carothers et al. 2006)
	8	L-Arginine	7.6	Mg 2+	(Famulok 1994)
	9	L-Citrulline	7.6	Mg 2+	(Famulok 1994)
	10	L-Arginine	7.6	Mg 2+	(Geiger et al. 1996)
	11	L-Tyrosine	7.4	Mg 2+	(Mannironi et al. 2000)
	12	L-Tryptophan 7.0	Mg 2+ , Ca 2+ , glycine	(Majerfeld and Yarus 2005)

Table 3 .

 3 In vitro selected ribozymes with ligation and cleavage activity

	No. Function	pH	Cofactor	Reference
	1	RNA ligation	7.4	Mg 2+	(Bartel and Szostak 1993)
	2	RNA ligation	7.4	Mg 2+	(Ekland et al. 1995)
	3	RNA ligation	7.4	Mg 2+	(Hager and Szostak 1997)
	4	RNA ligation	7.4	Mg 2+	(Teramoto et al. 2000)
	5	RNA ligation	4.0	Mg 2+	(Miyamoto et al. 2005)
	6	RNA ligation	7.5	Mg 2+	(Ohuchi et al. 2008)
	7	RNA ligation	7.5	Mg 2+	(Fujita et al. 2010)
	8	RNA ligation	8.0	Mg 2+	(Lie et al. 2016)
	9	RNA cleavage	7.5	Pb 2+	(Pan and Uhlenbeck 1992)
	10 RNA cleavage	8.1	MgOAc	(Williams et al. 1995)
	11 RNA cleavage	5.0	no	(Jayasena and Gold 1997)
	Two other main ribozyme families are the phosphorylating and aminoacylating ribozymes
	(Table 4). Phosphorylating ribozymes (Westheimer 1987) may promote enzymatic reactions
	such as RNA/DNA ligation and RNA degradation in vivo by adding phosphate group(s) to RNA
	targets. Previous in vitro selection have identified such ribozymes and shown the phos-
	phorylation for RNA polymerization

Table 4 .

 4 Ribozymes being able to catalyze phosphorylation and aminoacylation

	No. Function	pH	Cofactor	Reference
	RNA phosphorylation	7.4	Mg 2+ , Mn 2+ , ATP	(Lorsch and Szostak 1994)
	RNA phosphorylation	7.25 Mg 2+ , Ca 2+ , ATP	(Curtis and Bartel 2005)
	RNA phosphorylation	6.5	Mg 2+ , Ca 2+ , Mn 2+ , ATP	(Saran et al. 2005)
	RNA phosphorylation	7.5	Mg 2+ , Ca 2+ , Mn 2+ , Cu 2+ , ATP	(Biondi et al. 2013)
	RNA triphosphorylation 8.1	Mg 2+ , trimetaphosphate	(Moretti and Müller 2014)
	RNA triphosphorylation 8.3	Mg 2+ , trimetaphosphate	(Dolan et al. 2015)
	Acyl-transfer	7.3	Mg 2+ , Met-O-RNA	(Lohse and Szostak 1996)
	Acyl-transfer	7.4	Mg 2+ , Phe-AMP	(Jenne and Famulok 1998)
	RNA aminoacylation	7.0	Mg 2+ , Ca 2+ , Phe-AMP	(Illangasekare et al. 1995)
	RNA aminoacylation	8.0	Mg 2+ , Met-O-RNA	(Lee et al. 2000)
	RNA aminoacylation	7.5	Mg 2+ , Phe-CME/AMP/TE	(Saito et al. 2001)
	RNA aminoacylation	7.25 Ca 2+ , Phe-GMP	(Illangasekare and Yarus 1999)
	Acyl-transfer	7.0	Mg 2+ , Phe-p-KK13	(Xu et al. 2014)
	Peptide bond formation 7.4	Mg 2+ , Met-AMP	(Zhang and Cech 1998)
	Peptidylation	7.25 Mg 2+ , Ca 2+ , Phe-AMP	(M. Illangasekare 1999)
	Peptidylation	7.4	Mg 2+ , Met-AMP	(Sun et al. 2002)
	Amide bond formation	7.4	Mg 2+ , Ca 2+ , PheCoA	(Li and Huang 2005)
	Amino acid activation	4.0	Ca 2+	(Kumar and Yarus 2001)

Table 2

 2

		-1. Initial design of the RNA pool
	Name RNA Sequence (5'→3')
	N16 a GGN NNN NNN NNN NNN NNN
	...	...
	N45	GGN NNN NNN NNN ……… NNN NNN NNN NNN NNN

a different number indicates the length of the template Another possibility to generate 3'-end homogeneous transcripts is to add the sequence of the HDV self-cleaving ribozyme on the DNA template after the 3'-end. In that way, a clean and unique 3'-end can be generated after transcription. It however requires a PCR with extended primer PCR and extra gel purification procedures

[START_REF] Schürer | A universal method to produce in vitro transcripts with homogeneous 3' ends[END_REF][START_REF] Chumachenko | Rapid and Simple Ribozymic Aminoacylation Using Three Conserved Nucleotides[END_REF]

). Because our RNA pools are very short, these extra procedures would imply a significant loss of material during each purification step. In order to prevent transcripts with alternate sizes from invading the selection process, the terminal nucleotides of the sequences were set to very short conserved bases, either SCA or YCCA (Table

2-2

). During reverse transcription, by using a primer of the adapter with an overhang complementary to these nucleotides, most of these alternate transcripts could not undergo reverse transcription (Table 2-2, figure).

Table 2 -

 2

2. Template designed with conserved

Table 2

 2

	-4. Template design with 10 conserved nucleotides
	RNA Sequence (5'→3')
	GGC ACG ANN NNN ……… NNN NNN NNN UCR a
	GGC GAC GNN NNN ……… NNN NNN NNN UCR a
	a R=A or G

Table 2

 2

		-5. Final template design
	Name Sequence	n (nmol) Diversity Copies per species
	N19	GGCACGANNNNNNNNNUCA	0.1	2.6×10 5	2×10 8
	N23	GGCACGANNNNNNNNNNNNNUCA	0.1	6.7×10 7	1×10 6
	N30	GGCACGANNNNNNNNNNNNNNNNNNNNUCA	0.1	1.1×10 12 60
	N37	GGCACGANNNNNNNNNNNNNNNNNNNNNNNNNNNUCA 0.1	1.8×10 16 1

Table 2 -6.

 2

Table 2 -

 2 6.Protocol of incubation to promote potential ribozyme aminoacylation

	Components

.4 Selection procedure and controls: the OD-DO Assay 2.4.1 Sodium periodate oxidation

	Sodium periodate (NaIO4) oxidation has been widely used in organic chemistry for specifically
	oxidizing cis-diol groups into di-aldehyde group. Cis-diol groups are normally present on the
	3'-end of most RNA molecules. Under appropriate reaction conditions, periodate can eliminate
	the ribose five-membered ring up to 99% (Figure 2-9)

Table 2

 2

	-7. OD-DO assay
			RNA pellet
		OD a	DO a	D	NT
	NaOAc/HOAc (3M pH=5.2)	5 μl	0	0
	H2O	40 μl	43 μl	43 μl	50 μl
	NaIO4 sol. (0.1M) b	5 μl	0	0
	Borax buffer (pH=10.0)	0	7 μl	7 μl
		on ice 45 min c	42º C 1h	42º C 1h	on ice 1h
			Precipitation c
	NaOAc/HOAc	0	5 μl
	H2O	43 μl	40 μl
	NaIO4 sol. (0.5M) b	0	5 μl
	Borax buffer (pH=10.0)	7 μl	0
		42º C 1h	on ice 45 min c

Table 2 - 8 .

 28 The sequences of Class I adapters (Grey color indicates the restriction site).

	Name	Sequence (5'→3')
	Adapter K	5Phos/rUrUrUrUrGrArArGrArGrCrGrGrCrCrG
	Adapter L	5Phos/rUrUrUrUrCrArGrGrArUrArCrGrCrCrGrCrU/3InvdT/
	Adapter XL	5Phos/rUrUrUrUrCrArGrGrArUrArCrGrGrArGrUrUrArArCrU
		rUrUrGrCrArUrArGrGrCrGrArUrUrGrCrArA/3InvdT/
	Adapter M	5Phos/rUrUrUrUrCrArGrGrArUrArCrGrCrArGrUrCrUrArCrU
		rG/3InvdT/

Table 2

 2

	-9. Sequence of Adapter Hyb-XL
	Name	Sequence (5'→3')
	Adapter Hyb-XL	5Phos/rUrUrUrUrCrArGrGrArUrArCrUrCrGrCrArGrUrUr
		ArArCrUrCrGrGrCrArUrArGrGrCTTTTGCCTATGCCGAGTTAA
		CTGC

Table 2

 2

	-10. Sequences of Class III adapters
	Name	Sequence (5'→3')
	Adapter N	/5Phos/rUrUrUrUrGrUrGrGrArUrArCrGrCrArGrUrCrUrArCrUr
		GTTTTCAGTAGACTGCGTATCCACAAAATGG
	Adapter NR	/5Phos/rUrUrUrUrGrUrGrGrArUrArCrGrCrArGrUrCrUrArCrUr
		GTTTTCAGTAGACTGCGTATCCACAAAATGGR

Table 2

 2

	-11. Sequences of Class IV adapters
	Name	Sequence (5'→3')
	Adapter O	/5Phos/rUrUrUrUrUrCrCrCrArGrUrCrArGrCrUrGrUrCrUrArGrU
		TTTTACTAGACAGCTGACTGGG
	Adapter P	/5Phos/rArGrArArGrArGrCrCrGrUrUrArGrCrUrGrUrCrUrArGrU
		TTTTACTAGACAGCTAACGGCTC

This restriction site was selected to take advantage of the strong base paring interaction GGG/rCrCrC at the end of the duplex (Figure

2

-23), which minimizes fraying (

Table 2-11, red).

Table 3

 3

		-1. Sequence diversity of initial pool
	Name Diversity Initial amount Copy number of each unique sequence
	N19	2.6×10 5	0.025 nmol	~10 8
	N23	6.7×10 7	0.025 nmol	~10 6

Table 3 -

 3 2, A. Sequencing results of N19 pool after 1 st cycle selection Sequence Optimal secondary structure GGCACGAAGAAAAUUGUCA ((((..........)))).

Table 3 -

 3

	3. SELEX parameters during the selection cycles
		RNA Purification	Initial quantity (nmol)	RT-PCR cycles
	Library name Cycle 1-3 Cycle 4-6 or 7 Cycle 1-3 Cycle 4-6 or 7	Cycle 1	Cycle 4-6 or 7
	N19	Gel	HPLC	0.025	0.01	35 (50 µl x 5) 30 (50 µl x 2)
	N23	Gel	N/A	0.025	N/A	35 (50 µl x 5)	N/A
	N30	Gel	HPLC	0.025	0.01	35 (50 µl x 5) 30 (50 µl x 2)
	N37	Gel	HPLC	0.025	0.01	35 (50 µl x 5) 30 (50 µl x 2)

Table 3

 3

		-4. Sequencing result of library N19, 6 th round of selection
	No. Length Sequence (5'→3')	Copy # Secondary structure
	1	18nt	GGCACGAACAGCAUGUCA	1	((((.(.....).)))).

Table 3 -

 3 5. Sequencing result of library N23, 4 th round of selection

	No. Length Sequence	Copy # Secondary structure
	1	23nt	GGCACGACUAGACAGCCAACUCA	2

(((...........)))......

Table 3

 3

		-6. Sequencing result of library N30, 6 th round of selection
	No. Length Sequence	Copy # Secondary structure
	1	19nt	GGCACGACAGCACGCCUCA	2

(((..........)))...

Table 3

 3

	-7. Sequencing result of library N37, 6 th round of selection
	No. Length Sequence	Copy # Secondary structure
	23nt	GGCACGACUAGACAGCCAACUCA	1	(((...........)))......
	30nt	GGCACGAACAAACAUGUUACGCGCUCCUCA	1	(((.((((((....)))).)).))).....
		GGCACGAACCACGCUACCUGACCCACGUCA	1	(((.........)))...((((....))))
	34nt	GGCACGACCAGCGUUCCUCUGGACCCCUACGUCA	2	((.(((.....))).))....(((......))).
	36nt	GGCACGACCAGCGUUCCUCUGGACCCCUACGUCUCA	1	((.(((.....))).))...((((......))))..
	37nt	GGCACGACCAGCGUUCCCCUGGACCCCUACGUCUUCA	1

((.(((.....))).))...((((......))))... GGCACGACCGCCAUCGCCACUCCAACGUGGUCACUCA 1 (((......)))...(((((......)))))...... GGCACGACCACUCCCGUGGAACCACAGCAACGCCUCA 1 .......((((....))))..................

Table 3 -

 3 8. Sequencing result of library N37, 7 th round of selection

	No. Length Sequence

((((......)))).

Table 3

 3

		-9. Categories I to V
	Cat. Sequence	Copy # Secondary structure Library
		GGCACGACUAGCCAGCUCA	2	(((.......)))......	N19(6r)
	I	GGCACGACUAGCCAGCCCCGUCA	1	(((.......)))..........	N19(6r)
		GGCACGACUAGCCAGCCAACUCA	1	(((.......)))..........	N37(7r)
		GGCACGAACAGCAACGUCA	1	.((.......)).......	N19(6r)
	II	GGCACGAACAGCCAUCCGACUCA	1	(((.......)))..........	N19(6r)
		GGCACGAACAGCACGACUCA	1	.((.......))........	N30(6r)
		GGCACGACAGCACGCCUCA	1	(((..........)))...	N19(6r)
	III	GGCACGACAGCACGCCUCA	2	(((..........)))...	N30(6r)
		GGCACGACAGCACGCCUCA	1	(((..........)))...

Table 2 . 1 .

 21 Concentrations of total RNA recovered from OD, DO, D and NT treatments

		OD	DO	D	NT
	c (ng/µl)	399.2	356.1	415.4	409.5

Table 2

 2 GGG AGA GAG AGA GAA UUA CCC UCA CUA AAG GGA GGA GAA GCU UAU CCC AAGAUC CAA CUA CGA GCU UUU UAA CUG CAG CAA CUU UAA UAU ACG CUA UUG GAG CUG GAA UUU CCG CGG CUG CUG UUC UAG AG A Stock solution of the three spikes mixture at approximately equal molar concentration (1:0.94:0.85) was prepared, and the mass concentration of the solution was established by nanodrop (2269 ng/µl). This stock was diluted to 1/200 and 2 µl were added to each sample, corresponding to a ratio of approximately 5% of spike per RNA sample (in mass).

	.2. Spikes used for normalization
	RNAspike_25 (for 10-50 nt category)	Size: 25 nt	MW: 8172.9 g/mol
	GGG AGA GAG GGC UGC UGU UCU AGA G
	RNAspike_65 (for 50-100 nt category)	Size: 65 nt	MW: 20991.6 g/mol
	GGG AGA GAG AGA GAA UUA CCC UCU ACG CUA UUG GAG CUG GAA UUU CCG CGG
	CUG CUG UUC UAG AG
	RNAspike_25 (for 10-50 nt category)	Size: 25 nt	MW: 8172.9 g/mol

Table 2 .

 2 3: cDNA banks prepared by the Sequencing Platform Facility at Gif-sur-Yvette.

	Bank name	Inserts size (nt)	Index	Concentration (ng/µl)	Volume (µl)
	OD-A	100-250	16	13,6	18
	OD-B	50-100	17	6,37	18
	OD-C	10-50.	18	27	18
	DO -A	100-250	10	0,74	18
	DO -B	50-100	11	4,11	18
	DO-C	10-50.	12	21,9	18
	D-A	100-250	19	1,53	18
	D-B	50-100	20	7,78	18
	D-C	10-50.	21	27,9	18
	NT-A	100-250	13	3,36	18
	NT-B	50-100	14	8,49	18
	NT-C	10-50.	15	17,4	18

Table 2 .

 2 4. Mapping of sequencing reads onto E. coli and S. cerevisiae genomes

	Bank	Total number of	% mapping against E. coli BL21	% mapping against S. cerevisiae
	name	reads	genome	genome
	OD-A	18001500	87.8	11.1
	OD-B	24566899	11.2	83.1
	OD-C	23488573	24.5	79.7
	DO-A	12521884	79.3	17.3
	DO-B	19888979	14.2	73.3
	DO-C	21609403	26.7	80.1
	D-A	16934962	93.5	5.8
	D-B	20650043	13.6	80.3
	D-C	23694044	23.9	78.4
	NT-A	22409868	96.2	3.2
	NT-B	21615843	17.4	76.9
	NT-C	20763821	25.1	78.1

Table 2 .5. Spike normalization RNAspike_65

 2

	Treatment MetZ-MetW-MetV	Maximum	Sample	Normalization factor	MetZ-MetW-MetV
		maximum coverage	coverage (mc)	conc. (sc)	(mcmin/mc)*(sc/scmin)	normalized coverage
	OD	10726	78246	399.2	0.5655	6066,04
	DO	10729	59684	356.1	0.6614	7095,98
	D	6589	39474	415.4	1.1665	7686,24
	NT	7066	45216	409.5	1.0039	7093,73

Table 2 .

 2 6. cDNA banks and Illumina Index Tag of the 16 samples.During final PCR, various amounts (50, 100 and 200 ng) of the first PCR were used to establish the best amplification conditions. Altogether 20 cycles of PCR were programmed to verify the presence of products. Gel analysis (Figure

	Sample ID Insert size (bp)	Index Number	Index Sequence Volume Concentration
	B0-1(OD)	15-280	Index 1	CGTGAT	20 µl	40.5 ng/µl
	B0-2(DO)	15-280	Index 2	ACATCG	20 µl	35 ng/µl
	B0-3(D)	15-280	Index 3	GCCTAA	20 µl	56.8 ng/µl
	B0-4(NT)	15-280	Index 4	TGGTCA	20 µl	44.2 ng/µl
	B1-1(OD)	15-280	Index 5	CACTGT	20 µl	29.5 ng/µl
	B1-2(DO)	15-280	Index 6	ATTGGC	20 µl	40.8 ng/µl
	B1-3(D)	15-280	Index 7	GATCTG	20 µl	35.4 ng/µl
	B1-4(NT)	15-280	Index 8	TCAAGT	20 µl	27.2 ng/µl
	B2-1(OD)	15-280	Index 9	CTGATC	20 µl	38 ng/µl
	B2-2(DO)	15-280	Index 10	AAGCTA	20 µl	49.5 ng/µl
	B2-3(D)	15-280	Index 11	GTAGCC	20 µl	31.5 ng/µl
	B2-4(NT)	15-280	Index 12	TACAAG	20 µl	50.8 ng/µl
	B3-1(OD)	15-280	Index 13	TTGACT	20 µl	34 ng/µl
	B3-2(DO)	15-280	Index 14	GGAACT	20 µl	23.2 ng/µl
	B3-3(D)	15-280	Index 15	TGACAT	20 µl	31.6 ng/µl
	B3-4(NT)	15-280	Index 16	GGACGG	20 µl	21.5 ng/µl

  Cover all the tubes with aluminum films; Dissolved 5.4 mg NaIO4 in 250 μl water to prepare 0.1 M sodium-periodate solution; All the oxidations are performed in dark and on ice. After each oxidation, add 10 μl Glucose (0.5 M) and equilibrate for 15 min on ice. Then add 6 μl DTT before precipitation.

	Components	Volume			Final concentration
	RNA (around 5 μM)	(adjustable) 5 μl 0.25 μM (1-3cycle), 0.1 μM (remaining cycles)
	NaCl (1 M)	10 μl			100 mM
	KCl (1 M)	10 μl			100 mM
	Total	H2O fills up to 30 μl
	Incubate at 90°C for 3 min and cool down to 60°C
	CaCl2 (250 mM)	1 μl			2.5 mM
	MgCl2 (250 mM)	2 μl			5 mM
	MnCl2 (250 μM)	2 μl			5 μM
	Poly-lysine (0.01%)	2 μl
	Poly-arginine (0.01%)	2 μl
	Spermine (250 μM)	1 μl			2.5 μM
	aa Mix (42.5 mM)	40 μl			17 mM
		Mix well and chill on ice
	GTP (100 mM)	2 μl			2 mM
	ATP (100 mM)	2 μl			2 mM
		Mix well and avoid any precipitation
	NaOH (1 M)	3.5 μl			35 mM
					pH = 3.5
	Total	88 μl
		Incubate on ice overnight
	HEPES buffer (1 M, pH=7.0)	12.5 μl			pH 7.0
	Total	100.5 μl
		Incubate on ice for 2h and then precipitate
	3) OD-DO Assay
		OD	DO	D	NT
	NaOAc/HOAc (3M pH=5.2)	5 μl	0	0
	H2O	Up to 50 μl	Up to 50 μl	Up to 50 μl	Up to 50 μl
	NaIO4 sol. (0.1M)	5 μl	0	0
	Borax buffer (pH=10.0)	0	7 μl	7 μl
		on ice 45 min	42º C 1h	42º C 1h	on ice 1h
			Precipitation
	NaOAc/HOAc	0	5 μl
	H2O	Up to 50 μl	Up to 50 μl
	NaIO4 sol. (0.5M)	0	5 μl
	Borax buffer (pH=10)	7 μl	0
		42º C 1h	on ice 45 min
			Precipitation

treatment and spike adding (1 st sequencing)

   pellet in 88 µl H2O, 10X buffer Turbo ® DNase 10 µl, Turbo DNase (U/μl) 2 µl; Incubate 1h at 37°C then keep at 16°C overnight. Do Phenol/Chloroform extraction the next day. Measure the concentration of all the treated samples OD/DO/D/NT by Nano-drop. Add exactly the same mass percentage pre-mixed Spike-25/65/135 nt into 4 samples, 5% per sample. After spike adding, verify the final concentration. Run bioanalyzer to check the RNA quality. PCR to verify the DNase treatment efficiency by using the primers pairs tRNA-tyr5'/tRNA-tyr3'. PCR mix: RNA sample 1 µl, DreamTaq polymerase (ThermoFisher) 0.1 µl, 10X DreamTaq Green buffer 2.5 µl, dNTPs (5mM) 2 µl, primer Tyr forward and reverse (10 µM) 2 µl for each, H2O fills up to 25 µl. Run 30 cycles PCR (95°C 2 min; [95°C 20s, 55°C 20s, 72°C 20 s]; 72°C 2 min)

	DNase
		OD	DO	D	NT
			4 μg total RNA for each sample
	NaOAc/HOAc (3M pH=5.2)	5 μl	0	0
	H2O	Up to 50 μl	Up to 50 μl	Up to 50 μl	Up to 50 μl
	DMSO	2 μl	2 μl	2 μl	2 μl
	NaIO4 sol. (0.1M)	5 μl	0	0
	Borax buffer (pH=9.0)	0	7 μl	7 μl
		r.t. 30 min	42ºC 45 min	42ºC 45 min	on ice 45 min
			Precipitation
	NaOAc/HOAc	0	5 μl
	H2O	Up to 50 μl	Up to 50 μl
	DMSO	2 μl	2 μl
	NaIO4 sol. (0.5M)	0	5 μl
	Borax buffer (pH=9.0)	7 μl	0
		42ºC 45 min	r.t. 30 min
			Precipitation

DNase treatment and spike adding (2 nd sequencing)

  Re-suspend treated RNA (around 1~4µg) pellet in 88 µl H2O, 10X buffer Turbo ® DNase 10 µl, Turbo DNase (U/μl) 2 µl; Incubate 4h at 37°C then keep at 16°C overnight. Do Phenol/Chloroform extraction the next day. Measure the concentration of all the treated samples OD/DO/D/NT by Nano-drop. Add exactly the same mass percentage Spike-65nt into 4 samples, 2% per sample. After adding spike, verify the final concentration. Run 4% agarose gel at 135 voltages for 35 min to check the RNA quality. PCR to verify the DNase treatment efficiency by using the primers pairs tRNA-tyr5'/tRNA-tyr3'. PCR mix: RNA sample 1 µl, DreamTaq polymerase (ThermoFisher) 0.1 µl, 10X DreamTaq Green buffer 2.5 µl, dNTPs (5mM) 2 µl, primer Tyr forward and reverse (10 µM) 2 µl for each, H2O fills up to 25 µl. Run 30 cycles PCR (95°C 2 min; [95°C 20s, 55°C 20s, 72°C 20 s]x30; 72°C 2 min) RNA ligation OD, DO, D, NT treated RNA (1 µg per tube) 5 µl (adjustable), Pre-adenylated RNA 3' adapter (50 µM) 1 µl; Mix well and then place on PCR machine, denature at 70°C for 2 min then chill on ice immediately. Add 10x T4 RNA ligase 2 buffer 1 µl, DMSO 1 µl, BSA 1 µl, T4 RNA ligase 2 truncated (NEB) 1 µl. Incubate at 10°C overnight. The next day add 40 µl H2O then precipitation.

  ). Using Millipore 10K spin column clean the reaction solution (50 µl reaction solution + 450 µl water, spin 10 min; refill 450 µl water, spin 20 min, collect leftover). Precipitate overnight and suspend the pellet into 45 µl H2O and 5 µl TE buffer. 10X AccuPrime ™ buffer I 5 µl, Illumina RNA RT primer (10 µM) 1 µl, Reverse polyT primer (10 µM) 1 µl, cDNA template (50 ng) 5 µL, AccuPrime ™ Taq polymerase 1 µl; Run 10 to 18 cycles (depends) PCR; (94°C 2 min; [94°C 30 s, 41+1°C 40 s, 68°C 20 s] x 5; [94°C 30 s, 56°C 30 s, 68°C 20 s] x 13; 68°C 2 min).

	The first round PCR
	PCR Mix: H2O 37 µl,

With this adapter, ligation and RT-PCR works much better than in all our previous attempts. Unfortunately, we could not achieve a proper digestion with BmrI already at the end of the first round selection. We tried to optimize temperature, substrate concentration, incubation time, and despites all our efforts it seemed impossible to get clear digestion products (Figure 2-24).

Furthermore, the digested product was also very hard to recover by gel purification. The origin of this deficiency remained unclear, and we finally had to give up with this adapter and switch to Adapter P which contains a restriction site for the enzyme "EarI". EarI is a type II restriction endonuclease that enables to specifically cleave one nucleotide at 3'-end of the recognition sequence on one strand, and four nucleotides away from the 5'-end on the opposite strand [5'…CTCTTC(N)/(N4)…3'] ( [START_REF] Polisson | EarI, a restriction endonuclease from Enterobacter aerogenes which recognizes 5'CTCTTC3[END_REF].

N37(7r)

Categories I to V all have one stem loop and bear slightly different 3'-end tails. At first sight, these stem loops seem compatible with the existence of a catalytic site where ATP/GTP and an amino acid could bind, while the unstructured 3'-ends may possibly bind nearby. We established the distribution of the length of the single stranded 3' end in categories I -V (Figure

3-8).

Interestingly, even though these sequences were selected independently from different libraries, the length of their unstructured 3'-end tails is not very disparate: most of them are between 3 and 10 nt and the distribution shows a narrow profile centered around 6 nt (Figure 3-8, left). We also analyzed the length distribution of the 3' end tail within the IV category, which is the most abundant (Figure 3-8, right). There are two peaks at 4 nt and 6 nt, while no representative is found with a 3' tail of 5 nt. This phenomenon seemed reminiscent of a structural effect observed with the Yarus 29-nt ribozyme [START_REF] Lehmann | Efficiency of a self-aminoacylating ribozyme: effect of the length and base-composition of its 3' extension[END_REF]) -one nucleotide change could cause a significant drop in the ribozyme activity and thus affect its enrichment among the population. Categories VI and VII are presented in the table below:

N37(6r)

GGCACGACCAGCGUUCCUCUGGACCCCUACGUCUCA 1

((.(((.....))).))...((((......))))..

N37(6r)

GGCACGACCAGCGUUCCCCUGGACCCCUACGUCUUCA 1

((.(((.....))).))...((((......))))...

N37(6r)

The above results suggested to us that our selection strategy to isolate self-aminoacylating ribozymes was successful. An additional investigation during which oligonucleotides involved in our selection protocol were compared to the selected sequences however revealed an unexpected phenomenon: the most frequently selected 23-nt RNA has a sequence identical to a portion of the 3' adaptor-primer used in the RNA ligation step (Figure 3456789).

Experiment

Sample preparation

For this second round of deep-sequencing analysis, we decided to work with E. coli MG1655 instead of E. coli BL21 on suggestion of Philippe Bouloc (I2BC). E. coli MG1655 being a well-studied strand of E. coli, new results will be easier to relate to existing data.

Bank preparation

Following the new workflow shown in We collect the gel in a range from 35bp to 300bp and extract the oligonucleotides with a "crush-and-soak" procedure (Supporting information, Gel purification).

Given the size of the 3' adapter (21 nt), purified cDNAs correspond to transcripts from ~15 to ~280 nt long. with PCR condition, we then choose to do a polyadenylation [START_REF] Gellert | HELIX FORMATION BY GUANYLIC ACID[END_REF][START_REF] Rhodes | G-quadruplexes and their regulatory roles in biology[END_REF][START_REF] Tateishi-Karimata | G-quadruplexes with tetraethylene glycol-modified deoxythymidines are resistant to nucleases and inhibit HIV-1 reverse transcriptase[END_REF].

Following the guidelines of the TdT protocol (NEB, M0315S), the incubation conditions were adjusted so as to achieve the addition of ~10 to 30 adenosine residues: each total RNA sample was incubated 45 min at 37º C with 150 µM ATP (Supporting information, TdT tailing).

PCR amplification

A first PCR reaction of all samples (17 cycles with an annealing temperature gradient from 41ºC to 46ºC during the 5 first cycles; T(annealing) = 56ºC from cycle 6 to 17) was achieved to

Analysis of tRNA reads with OD, DO, D and NT samples

An overview of the signals of four representative tRNA species (tyrosine-and lysine-tRNA) was achieved while collecting all the reads matching a 20-nt request corresponding to the first 20 nucleotides from the 3'-end of each tRNA. Because of the amount of sequencing errors, very few reads did match the request. This number could be increased while reducing the size of the request, consistent with the high number of errors.

Since the mapping of these reads on the E. coli genome could not provide any clear information, we decided to only collect some statistical properties from them. Although the maximum number of reads is only of a few tens in each case, the observed distribution already reveals a few useful observations:

1 -It can be seen that DO samples have much fewer reads compared with the other three conditions, as expected.

2 -An examination of the tRNA Tyr and tRNA Lys reads shows the presence of a peak at 21, 47 nt (for tRNA Tyr ) and 29 nt (for tRNA Lys ), which correspond to base modification at that position (Figure 2.19). This result suggests that our sequencing strategy is promising.

Improved protocol New Illumina universal primer

Newly designed forward adapter, with universal forward adaptor comprising a random NNNNN track at the end of the adapter, and before the polyT track which will allow sequencing machine to calibrate the signal at the beginning of sequencing process:

PCR improvement to eliminate primer-dimer contamination

In most of the sequencing bank preparation protocols, especially small RNA-seq, primerdimer removal is a critical step. If the RNA in-put is rather low, both beads binding and gel purification are required [START_REF] Quail | Improved Protocols for the Illumina Genome Analyzer Sequencing System[END_REF][START_REF] Kumar | A High-Throughput Method for Illumina RNA-Seq Library Preparation[END_REF].

Primer-dimer contamination has also been revealed from our deep-sequencing results.

Even though we did do the gel purification very carefully after reverse transcription, however, it is impossible to remove all the primer dimers. Possibly, total RNA could embed very few single stranded adapter and thus cause the downstream experiment generating primer-dimer byproduct. From the PCR results we can see that this contamination is not a significant problem for OD, D and NT samples, while since the DO experiment is negative selection, the leftover RNA target will be much less than the primer-dimer which will cause the deep-sequencing data was dominated by the primer-dimer signal (Figure 2.20, left). This contamination will not only take up the quantity of meaningful reads, but also affect the spike normalization.

In order to avoid this contamination, we tried a new PCR strategy. Since we are interested in aminoacylated tRNA, we here decide to use a modified Illumina RT primer carrying three bases "TGG" at the 3'-end which is overhanging with the tRNA's CCA-ending. With this new primer, primer-dimer is not visible even after 35 cycles PCR (Figure 2.20, right). Meanwhile, we also changed the spike to a fraction of human 18S RNA containing a CCA ending (5'-

GACGAUCAGAUACCGUCGUAGUUCCGACCA-3').

T4 RNA ligation RNA pellet, Adaptor pellet (0.1 nmol), H2O 14 μl, 10X T4 RNA Ligase Buffer 4 μl, T4 RNA Ligase (5 U/μl) 2 μl (ThermoFisher), PEG8000 (50%) 20 μl; Mix well and incubate at 10°C overnight. Then precipitate to remove the reaction buffer.

Reverse transcription

Gel purification

Use 30K Amicon ® Ultra-0.5 Centrifugal Filter Millipore spin column to concentrate 5 tubes PCR products and load on 4% agarose gel with ethidium bromide. Run the gel at 135 voltages for 30 min in 0.5X TAE electrophoresis running buffer. Cut the gel at expected size and chop it into small pieces. Soak the gel into 450 μl H2O + 50 μl TE Buffer (Sigma) and shake at 20ºC overnight. Precipitate to collect DNA pellet. With newly designed SELEX protocol, we aim to uncover small ribozymes of the order of 20 nucleotides that could catalyze both amino acid activation and transesterification. Although molecules catalyzing either reaction have been identified, no existing ribozyme could use free amino acids and activating cofactor(s) as substrates for 3' esterification in a single reactional context.

Re-amplification

The selection of active molecules in a SELEX procedure requires the presence of constant tracks on both ends of the sequences constituing the initial random pools. These tracks are required for PCR, but they impose the burden to the identification of ribozymes. We present an optimized protocol that significantly minimizes the size of these constant tracks. And our newly design protocol is very specific for the selection of 3'-end aminoacylated RNA. We performed 6 to 7 cycles of selection with different pools, and observed an enrichement with specific sequences. Although some experiments did reveal a possible activity, no activity could be so far confirmed with specific sequences.

A similar protocol was also applied in a parallel study to identify aminoacyl RNA from total RNA in E.coli. Our goal is to possibly identify new classes of aminoacyl RNA by deep sequencing. Using tRNA to validate our protocol, we realized that a standard RNAseq procedure couldn't work due to the presence of modified bases. We established a new method for bank preparation to identify any sequence aminoacylated at the 3' end. Ultimately, this new approach will allow us to study the level of aminoacylation of any sequence present in total RNA.