Pascal Röthlisberger

Fabienne Levi-Acobas

Christian J Leumann

Marcel Hollenstein
email: marcel.hollenstein@pasteur.fr

Enzymatic synthesis of biphenyl-DNA oligonucleotides

Keywords:

The incorporation of nucleotides equipped with C-glycosidic aromatic nucleobases into DNA and RNA is an alluring strategy for a number of practical applications including fluorescent labelling of oligonucleotides, expansion of the genetic alphabet for the generation of aptamers and semi-synthetic organisms, or the modulation of excess electron transfer within DNA. However, the generation of C-nucleoside containing oligonucleotides relies mainly on solidphase synthesis which is quite labor intensive and restricted to short sequences. Here, we explore the possibility of constructing biphenyl-modified DNA sequences using enzymatic synthesis. The presence of multiple biphenyl-units or biphenyl residues modified with electron donors and acceptors permits the incorporation of a single dBphMP nucleotide. Moreover, templates with multiple abasic sites enable the incorporation of up to two dBphMP nucleotides, while TdTmediated tailing reactions produce single-stranded DNA oligonucleotides with four biphenyl residues appended at the 3'-end.

Introduction

DNA is a fascinating biopolymer that mainly serves as the repository of the genetic information. The well-known double helical structure adopted by DNA arises through specific Watson-Crick interactions and packing forces between the four canonical nucleobases A, C, G, and T. However, a combination of organic chemistry and polymerase engineering have not only permitted to broaden the horizon of applications of DNA to fields that markedly deviate from its natural role [START_REF] Röthlisberger | Aptamer Chemistry[END_REF][START_REF] Taylor | Catalysts from synthetic genetic polymers[END_REF][START_REF] Hocek | Enzymatic Synthesis of Base-Functionalized Nucleic Acids for Sensing, Cross-linking, and Modulation of Protein-DNA Binding and Transcription[END_REF][START_REF] Hollenstein | Nucleic acid enzymes based on functionalized nucleosides[END_REF][START_REF] Goyenvalle | Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers[END_REF][START_REF] Dunn | Analysis of aptamer discovery and technology[END_REF][START_REF] Gasse | Modulation of BACE1 Activity by Chemically Modified Aptamers[END_REF][START_REF] Pinheiro | Synthetic Genetic Polymers Capable of Heredity and Evolution[END_REF][START_REF] Flamme | Chemical methods for the modification of RNA[END_REF][START_REF] Eremeeva | Highly stable hexitol based XNA aptamers targeting the vascular endothelial growth factor[END_REF][START_REF] Chidchob | Spatial Presentation of Cholesterol Units on a DNA Cube as a Determinant of Membrane Protein-Mimicking Functions[END_REF][START_REF] Bujold | DNA Nanostructures at the Interface with Biology[END_REF] but also to expand the genetic alphabet by one [START_REF] Kimoto | Generation of high-affinity DNA aptamers using an expanded genetic alphabet[END_REF][START_REF] Zhang | A semisynthetic organism that stores and retrieves increased genetic information[END_REF][START_REF] Malyshev | A semi-synthetic organism with an expanded genetic alphabet[END_REF][START_REF] Malyshev | The Expanded Genetic Alphabet[END_REF] or multiple [START_REF] Hoshika | Hachimoji DNA and RNA: A genetic system with eight building blocks[END_REF] orthogonal, artificial base pairs. Unnatural base pairs (UBPs) that have been used in this context mainly consist of large aromatic residues that interact via hydrophobic and packing forces, [START_REF] Feldman | Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism[END_REF] nucleobases with alternative hydrogen bond patterns, [START_REF] Hoshika | Hachimoji DNA and RNA: A genetic system with eight building blocks[END_REF] and nucleoside analogs capable of interacting through shape complementarity. [START_REF] Singh | Snapshots of an evolved DNA polymerase pre-and postincorporation of an unnatural nucleotide[END_REF][START_REF] Kimoto | Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA[END_REF] More recently, artificial metal base pairs have been proposed as alternative scaffolds for the construction of UBPs. [START_REF] Müller | Nucleic acid duplexes with metal-mediated base pairs and their structures[END_REF][START_REF] Kobayashi | Enzymatic synthesis of ligand-bearing DNAs for metal-mediated base pairing utilising a template-independent polymerase[END_REF][START_REF] Kim | Polymerase Recognition of a Watson-Crick-Like Metal-Mediated Base Pair: Purine-2,6-Dicarboxylate center dot Copper(II)center dot Pyridine[END_REF][START_REF] Kaul | Reversible bond formation enables the replication and amplification of a crosslinking salen complex as an orthogonal base pair[END_REF][START_REF] Levi-Acobas | On the Enzymatic Formation of Metal Base Pairs with Thiolated and pK(a)-Perturbed Nucleotides[END_REF][START_REF] Röthlisberger | Towards the enzymatic formation of artificial metal base pairs with a carboxy-imidazole-modified nucleotide[END_REF][START_REF] Röthlisberger | On the enzymatic incorporation of an imidazole nucleotide into DNA[END_REF][START_REF] Takezawa | Enzymatic Synthesis of Cu(II)-Responsive Deoxyribozymes through Polymerase Incorporation of Artificial Ligand-Type Nucleotides[END_REF] Such an expansion of the genetic alphabet has important practical repercussions since semisynthetic organisms capable of replicating DNA containing artificial nucleotides [START_REF] Zhang | A semisynthetic organism engineered for the stable expansion of the genetic alphabet[END_REF] and even transcribing modified mRNA into proteins [START_REF] Zhang | A semisynthetic organism that stores and retrieves increased genetic information[END_REF] could be created and aptamers with enhanced binding properties were identified. [START_REF] Kimoto | Generation of high-affinity DNA aptamers using an expanded genetic alphabet[END_REF][START_REF] Kimoto | Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA[END_REF][START_REF] Zhang | Evolution of Functional Six-Nucleotide DNA[END_REF][START_REF] Zhang | Aptamers against Cells Overexpressing Glypican 3 from Expanded Genetic Systems Combined with Cell Engineering and Laboratory Evolution[END_REF] In this context, the zipper-like interstrand stacking motif (Fig. 1A) is an alternative recognition motif based on the interaction of Cnucleotides equipped with bipyridine (Bipy) or biphenyl (dBph) moieties within a DNA duplex (Fig. 1B). [START_REF] Brotschi | Bipyridyl-and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking[END_REF][START_REF] Brotschi | A stable DNA duplex containing a non-hydrogen-bonding and non-shape-complementary base couple: Interstrand stacking as the stability determining factor[END_REF][START_REF] Brotschi | DNA with hydrophobic base substitutes: A stable, zipperlike recognition motif based on interstrand-stacking interactions[END_REF] The inclusion of four biphenyl base pairs in opposing positions in short DNA duplexes gave rise to important thermal stabilities (T m of up to 4.4°C per base pair). [START_REF] Brotschi | DNA with hydrophobic base substitutes: A stable, zipperlike recognition motif based on interstrand-stacking interactions[END_REF] Interestingly, up to 7 consecutive biphenyl base pairs can be incorporated into DNA without causing any decrease in duplex stability [START_REF] Brotschi | Bipyridyl-and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking[END_REF] and an NMR investigation concluded that stacking interactions were the main driving force behind the impressive stability of these UBPs. [START_REF] Johar | Solution structure of a DNA duplex containing a biphenyl pair[END_REF] The zipper-like motif thus appears to be an alluring candidate for applications such as electron transport through nucleic acids [START_REF] Roethlisberger | Modulation of Excess Electron Transfer through LUMO Gradients in DNA Containing Phenanthrenyl Base Surrogates[END_REF][START_REF] Frommer | Preparation and characterization of pyrene modified uridine derivatives as potential electron donors in RNA[END_REF][START_REF] Antusch | Elucidation of the Dexter-Type Energy Transfer in DNA by Thymine-Thymine Dimer Formation Using Photosensitizers as Artificial Nucleosides[END_REF] or an expansion of the genetic code. [START_REF] Brotschi | RNA duplexes with biphenyl substituents as base replacements are less stable than DNA duplexes[END_REF] Besides biphenylmodified DNA, other C-nucleosides equipped with aromatic residues play central roles in numerous applications including the induction of important conformational transitions, [START_REF] Nakamura | Controlling Pyrene Association in DNA Duplexes by B-to Z-DNA Transitions[END_REF] relaying electron transport through duplexes, [START_REF] Frommer | Preparation and characterization of pyrene modified uridine derivatives as potential electron donors in RNA[END_REF][START_REF] Antusch | Elucidation of the Dexter-Type Energy Transfer in DNA by Thymine-Thymine Dimer Formation Using Photosensitizers as Artificial Nucleosides[END_REF][START_REF] Roethlisberger | Enhancement of Excess Electron Transfer Efficiency in DNA Containing a Phenothiazine Donor and Multiple Stable Phenanthrenyl Base Pairs[END_REF][START_REF] Singleton | Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling[END_REF][START_REF] Batzner | Acceleration of Long-Range Photoinduced Electron Transfer through DNA by Hydroxyquinolines as Artificial Base Pairs[END_REF] assisting the homogenous assembly of aromatic carbon layers, [START_REF] Chan | Luminescent Carbon Dot Mimics Assembled on DNA[END_REF] or labeling of oligonucleotides with fluorophores. [START_REF] Wilson | Fluorescent Probes of DNA Repair[END_REF][START_REF] Wilson | Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses[END_REF][START_REF] Nakamura | Highly ordered pyrene pi-stacks on an RNA duplex display static excimer fluorescence[END_REF][START_REF] Rovira | Expanding a fluorescent RNA alphabet: synthesis, photophysics and utility of isothiazole-derived purine nucleoside surrogates[END_REF][START_REF] Xu | Fluorescent nucleobases as tools for studying DNA and RNA[END_REF] However, solidphase synthesis of these analogs and biphenyl-DNA particularly is quite labor intensive and often complicated by the reduced solubility and the tendency to aggregate. Therefore, alternative methods such as enzymatic synthesis of dBph containing

A R T I C L E I N F O A B S T R A C T
oligonucleotides would be highly desirable. [START_REF] Brotschi | Bipyridyl-and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking[END_REF] Here, we explored the possibility of using enzymatic synthesis to generate oligonucleotides containing multiple dBph nucleotides. To this end, we have investigated whether 1. the formation of a zipper-like interstrand stacking motif could favor the successive incorporation of dBph nucleotides during primer extension (PEX) reactions and 2. the presence of multiple abasic sites on a template strand could be used to trigger the incorporation of multiple C-nucleotides into DNA. In this context, we demonstrate that the presence of three dBph residues on the template permits the incorporation of one modified nucleotide into DNA and that the introduction of abasic sites can be used to generate oligonucleotides containing up to two modified dBph analogs. We also show that the appendage of electron donors and acceptors on the distal ring of the biphenyl moiety can be used to improve the efficiency of enzymatic synthesis of DNA modified with aromatic C-nucleotides. Taken together, these results show that the enzymatic synthesis of oligonucleotides containing biphenyl C-nucleotide analogs is possible and could be extended to other zipper-like motifs [START_REF] Baumstark | Fluorescent hydrophobic zippers inside duplex DNA: Interstrand stacking of perylene-3,4 : 9,10-tetracarboxylic acid bisimides as artificial DNA base dyes[END_REF][START_REF] Nguyen | Duplex Stabilization and Energy Transfer in Zipper Porphyrin-DNA[END_REF][START_REF] Xiang | Role of electrostatic complementarity between perylenediimide and porphyrin in highly stabilized GNA[END_REF][START_REF] Meggers | Synthesis and Properties of the Simplified Nucleic Acid Glycol Nucleic Acid[END_REF][START_REF] Nakamura | Chromophore Arrays Constructed in the Major Groove of DNA Duplexes Using a Post-Synthetic Strategy[END_REF] and potentially for the construction of randomized libraries containing UBPs for SELEX experiments. [START_REF] Zhang | Evolution of Functional Six-Nucleotide DNA[END_REF][START_REF] Dien | Progress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic Alphabet[END_REF][START_REF] Betz | Structural Basis for Expansion of the Genetic Alphabet with an Artificial Nucleobase Pair[END_REF][START_REF] Hamashima | Creation of unnatural base pairs for genetic alphabet expansion toward synthetic xenobiology[END_REF] Figure 1. A) and B) details of an X-ray structure of an interstrand-stacking zipper motif (red) adapted from reference [START_REF] Roethlisberger | X-ray structure of a lectinbound DNA duplex containing an unnatural phenanthrenyl pair[END_REF] and C) chemical structure of dBphTP 1.

Results and discussion

Synthesis of the triphosphate and modified oligonucleotides

The synthesis of the modified nucleotide 1 proceeded by application of standard protocols (Scheme 1). [START_REF] Hocek | Enzymatic Synthesis of Base-Functionalized Nucleic Acids for Sensing, Cross-linking, and Modulation of Protein-DNA Binding and Transcription[END_REF][START_REF] Flamme | Chemical methods for the modification of RNA[END_REF] Briefly, nucleoside 2 [START_REF] Brotschi | Bipyridyl-and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking[END_REF][START_REF] Brotschi | DNA with hydrophobic base substitutes: A stable, zipperlike recognition motif based on interstrand-stacking interactions[END_REF] underwent a sequence of protecting group rearrangements which involved acetylation followed by detritylation. The resulting suitably protected nucleoside analog 4 was then triphosphorylated by application of the Ludwig-Eckstein conditions [START_REF] Ludwig | Rapid and efficient synthesis of nucleoside 5'-0-(1-thiotriphosphates), 5'-triphosphates and 2',3'cyclophosphorothioates using 2-chloro-4H-1,3,2benzodioxaphosphorin-4-one[END_REF] which after anion exchange HPLC (IE-HPLC) afforded dBphTP 1 in good yields (48%). The corresponding phosphoramidite building block for solid-phase DNA synthesis was obtained by application of a known protocol. [START_REF] Brotschi | Bipyridyl-and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking[END_REF] Scheme 1. Synthesis of nucleoside triphosphate dBphTP 1. Reagents and conditions: i) Ac2O, Et3N, DMAP, pyridine, rt, 2.5h, 95%; ii) DCA, DCM, rt, 30 min, 81%; iii) a) 2-chloro-1,3,2-benzodioxaphosphorin-4-one, pyridine, dioxane, rt, 45 min; (b) (nBu3NH)2H2P2O7, DMF, nBu3N, rt, 45 min; (c) I2, pyridine, H2O, rt, 30 min; (d) NH3(aq.), rt, 1.5 h, 48% (4 steps).

Natural and modified oligonucleotides were either purchased or prepared according to standard protocols for automated DNA synthesis on a 1 μmol scale in the trityl-off mode, respectively (see Table 1 andESI). [START_REF] Brotschi | Bipyridyl-and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking[END_REF][START_REF] Brotschi | DNA with hydrophobic base substitutes: A stable, zipperlike recognition motif based on interstrand-stacking interactions[END_REF] The modified nucleotides were introduced via the corresponding phosphoramidites (5-11, Figure 2) obtained as described previously. [START_REF] Zahn | Synthesis of functionalized biphenyl-Cnucleosides and their incorporation into oligodeoxynucleotides[END_REF][START_REF] Zahn | Recognition properties of donor-and acceptor-modified biphenyl-DNA[END_REF][START_REF] Röthlisberger | Incorporation of a minimal nucleotide into DNA[END_REF]

Evaluation of enzymatic synthesis via a zipper-like interstrand stacking motif

With dBphTP 1 at hand, we proceeded to evaluate the possibility of incorporating this C-nucleotide into DNA by enzymatic synthesis and eventually construct a zipper-like interstrand stacking motif. Therefore, we performed PEX reactions using different polymerases (Phusion, Hemo KlenTaq, Taq, Bst, Q5 DNA polymerase, Therminator, Vent (exo -), Sulfolobus DNA polymerase IV (Dpo4), Deep Vent, Phi29, and the Klenow fragment of DNA polymerase I exo -(Kf exo -)) along with dBphTP 1 as only substrate. We first evaluated the incorporation efficiency using a 24-nucleotide long template T1 (Table 1) containing a single dBph unit immediately downstream of the end of the 18-mer primer P1. [START_REF] Röthlisberger | On the enzymatic incorporation of an imidazole nucleotide into DNA[END_REF] This initial screen revealed that the modified nucleoside triphosphate was not readily accepted as a substrate since no elongation of the primer was observed (Fig. S1A). A similar observation was made with each of the natural dNTPs (data not shown) as well as with the abasic site nucleotide dTP 11 even though this analog was shown to be readily incorporated opposite templating dA units (Fig. S1B). [START_REF] Röthlisberger | Incorporation of a minimal nucleotide into DNA[END_REF] We hypothesized that this poor incorporation efficiency mirrors the trend observed in thermal stability of duplexes where a single dBph-dBph base pair caused a significant decrease in T m . [START_REF] Brotschi | Bipyridyl-and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking[END_REF] Hence, we assessed whether the presence of multiple dBph units on the template would spur the enzymatic formation of the zipper-like motif. We thus carried out PEX reactions with template T3 that contains three consecutive modified nucleotides immediately downstream of the end of primer P1 (Fig. 3). This analysis reveals that the polymerase Dpo4 incorporated a single dBphMP opposite a modified nucleotide in moderate yields (~50%) while other polymerases such as Hemo KlenTaq and Bst only poorly recognized the modified nucleotide as a substrate since only ~10% of the elongated primer was formed which is sometimes accompanied by exonucleolytic degradation of the primer (see e.g. lanes 1 and 6). A similar trend but with different polymerases was observed with dTP 11 since the n+1 product formed in ~50% yield with Bst and ~10% with Hemo KlenTaq (Fig. 3).

Effect of electron-donating and -withdrawing groups on enzymatic DNA synthesis

After a first evaluation of the possibility of an enzymatic construction of a zipper-like motif using the C-nucleoside analog dBph, we next questioned whether a modulation of the  electron density on the biphenyl moieties could have an impact on the incorporation efficiency of the modified nucleotide. Indeed, the inclusion of electron-withdrawing groups on the aromatic system was shown to significantly increase the T m value of the resulting duplexes while the presence of donating groups led to thermoneutral duplexes (mixed duplexes containing acceptor and donor moieties also led to increased T m values). [START_REF] Zahn | Recognition properties of donor-and acceptor-modified biphenyl-DNA[END_REF] In order to evaluate whether the nature as well as the position of substituents on the biphenyl moiety could favor the incorporation of a single dBph moiety into DNA we synthetized templates T4-9 that contain single biphenyl-nucleosides equipped with OMe, NH 2 , and NO 2 units in the distal ring in both para and meta orientations (Fig. 2 and Table 1). PEX reactions were carried out with primer P1, dBphTP, and various DNA polymerases (Fig. 4 and5). This analysis clearly reveals that various polymerases including Dpo4 and Kf exo -readily incorporate the modified nucleotide opposite biphenyl units equipped with a para-amino unit (template T3; Fig. 4A and Table S1). In addition, PEX reactions conducted with templates containing meta-nitro and para-methoxy modifications (T8 (Fig. 5C) and T5 (Fig. 4B), respectively) led to the formation of the expected n+1 product albeit in lesser yields and with fewer polymerases. Hence, the presence of electron-donors in the para-position or electronwithdrawing groups in the meta-position of the distal ring appears to be a means to improve the incorporation efficiency of the modified dBph nucleotide into DNA. On the other hand, enzymatic incorporation of the modified nucleotide was markedly reduced when the substitution pattern was reversed i.e. with electron-donors in the meta-position and electronwithdrawing groups in the para-position (see Fig. 4C and Fig. 5A and B and Table S1). Notwithstanding, partial formation of the n+1 extended primer product was formed in the presence of these templates which is not the case with the unmodified Bphcontaining template T1. The PEX reactions carried out with Therminator also seem to follow this trend even though partial exonucleolytic degradation of the primer can be observed. (Fig. 4 and Fig. 5) .

Enzymatic construction of oligonucleotides with multiple dBph units

Nucleotides equipped with non-hydrogen bonding aromatic moieties have been shown to be incorporated efficiently by DNA polymerases opposite abasic sites. [START_REF] Matray | A specific partner for abasic damage in DNA[END_REF][START_REF] Nakano | Conformational changes of the phenyl and naphthyl isocyanate-DNA adducts during DNA replication and by minor groove binding molecules[END_REF][START_REF] Motea | Insights into the Roles of Desolvation and pi-Electron Interactions during DNA Polymerization[END_REF] Since solid-phase synthesis of Bph-containing oligonucleotides is rather labor intensive, we hypothesized that such sequences could be constructed by the sequential enzymatic incorporation of modified nucleotides opposite abasic sites in PEX reactions. To do so, we synthesized three different templates containing one (T9), three (T10), or five (T11) consecutive abasic sites (Table 1). Gel analysis of PEX reactions with dBphTP revealed that the n+1 product was formed in good yields with a few DNA polymerases irrespective of the nature of the template (Fig. 6 and ESI). During reactions with template T9, a single dBphMP was readily incorporated opposite an abasic site by Therminator, Dpo4, and Kf exo -, while the PEX reactions with all other polymerases either led to little product formation, partial degradation of the primer, or no incorporation event (Fig. S2A and Fig. 6). Importantly, after the installation of such a dBph- pair, enzymatic DNA synthesis could be resumed by Therminator upon addition of natural nucleoside triphosphates (Fig. S3). Therefore, we propose a two-step protocol for the synthesis of any sequence containing a single dBphMP unit: 1. PEX reaction with Therminator, Dpo4, or Kf exo -and dBphTP on a template containing an abasic site 2. natural DNA synthesis after the installation of the dBph- pair with dNTPs and Therminator. Such a protocol has recently been applied for the synthesis of modified DNAzymes using metal base pairs [START_REF] Takezawa | Enzymatic Synthesis of Cu(II)-Responsive Deoxyribozymes through Polymerase Incorporation of Artificial Ligand-Type Nucleotides[END_REF] and DNA duplexes containing artificial silver base pairs. [START_REF] Flamme | Enzymatic Formation of an Artificial Base Pair Using a Modified Adenine Nucleoside Triphosphate[END_REF] In the former, a modified nucleotide was installed opposite natural dA and dT nucleotides and enzymatic synthesis was resumed with a different polymerase (Dpo4) while in the latter, a modified purine nucleotide was used to form an artificial silver base pair and natural DNA synthesis was resumed with Therminator. Our novel strategy mainly differs from these two first examples by the nature of the templating nucleotide (i.e. abasic sites) employed to trigger the incorporation of a C-nucleotide analog into DNA.

In the context of templates containing multiple abasic sites, efficient incorporation of a single dBph nucleotide was observed but no products corresponding to primer extended by multiple dBph units was formed (Fig. S2B). However, given that some of the polymerases displayed a high propensity at incorporating the modified nucleotide (high yields of product formation and no exonucleolytic degradation of the primer), we questioned whether conditions could be met that would enable the incorporation of multiple dBph units. We thus performed PEX reactions with increasing concentrations of modified nucleotide and by supplementing the reaction mixtures with 1 mM Mn 2+ which is known to relax the substrate specificity of DNA polymerases (Fig. 6). [START_REF] Horhota | Kinetic analysis of an efficient DNAdependent TNA polymerase[END_REF][START_REF] Tabor | Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage-t7 DNApolymerase and escherichia-coli DNA-polymerase-i[END_REF][START_REF] Chang | Human DNA Polymerase mu Can Use a Noncanonical Mechanism for Multiple Mn2+-Mediated Functions[END_REF][START_REF] Vichier-Guerre | 2'-Deoxyribonucleoside 5'-triphosphates bearing 4-phenyl and 4pyrimidinyl imidazoles as DNA polymerase substrates[END_REF] Variation of both parameters indeed resulted in higher incorporation efficiencies of dBphMP and the formation of the n+2 product (in yields up to ~30%), particularly with the polymerase Dpo4 and additional Mn 2+ . However, enzymatic synthesis of modified DNA appears to stall after the incorporation of two dBph unit since no n+3 product formation could be observed regardless of the nature of the template. For comparison, we also evaluated the incorporation efficiency of natural dATP on templates containing multiple consecutive abasic sites. In PEX reactions with template T10, Dpo4 efficiently incorporated two dAMP units and the n+3 product could be observed albeit in poor yields (<10%; Fig. 6A). When template T11 was used instead, Dpo4 produced a distribution of products while Therminator led to the formation of the n+2 and n+3 products. Thus, extension of primers with dAMP appears to be more efficient than with dBphTP but is nonetheless impaired by the presence of multiple abasic sites. Lastly, we evaluated the possibility of generating oligonucleotides equipped with dBph tails. To do so, we employed the X family polymerase terminal deoxynucleotidyl transferase (TdT) which incorporates nucleotides at the 3'termini of ssDNA oligonucleotides in a template-independent manner. [START_REF] Sarac | Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids[END_REF] Tailing reactions were carried out under different experimental conditions where the nature of the metal cofactors (Mn 2+ , Mg 2+ , and Co 2+ ), the influence of the initiator (i.e. primer) concentrations (2 and 20 µM) and the reaction time on the tailing reactions were evaluated (Fig. 7). When both Mg 2+ and Co 2+ were used as cofactors with low initiator concentrations, efficient tailing reactions were observed with the incorporation of up to four dBph units. On the other hand, when Mn 2+ was used as a cofactor, the tailing reactions were not as efficient since the polymerase seemed to stall after the addition of three modified nucleotides. The order of preference in terms of metal cofactors thus follows the trend often observed with modified nucleotides, i.e. Co 2+ ≈ Mg 2+ > Mn 2+ . [START_REF] Sarac | Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids[END_REF] Expectedly, decreasing the monomer/initiator ratio negatively impacted the TdT-mediated polymerization reactions. [START_REF] Tang | High-Molecular-Weight Polynucleotides by Transferase-Catalyzed Living Chain-Growth Polycondensation[END_REF] The appendage of four Cnucleotides on ssDNA primers during TdT-mediated tailing reactions appears to be a general upper limit for such analogs since similar results had been reported with pyrene-modified nucleotides. [START_REF] Hollenstein | Polymerase incorporation of pyrene-nucleoside triphosphates[END_REF][START_REF] Cho | Enzymatic synthesis of fluorescent oligomers assembled on a DNA backbone[END_REF]

Conclusions

The presence of C-nucleosides equipped with aromatic moieties in DNA can be used in a broad range of applications. However, while solid-phase synthesis permits the introduction of nucleoside analogs into DNA and RNA sequences, this method is restricted to rather short sequences and to simple modification patterns. Alternative protocols for the synthesis of oligonucleotides containing aromatic C-nucleotides are thus in dire need. [START_REF] Vasilyeva | DNA fluorescent labeling with naphtho 1,2,3-cd indol-6(2H)-one for investigation of protein-DNA interactions[END_REF] Enzymatic synthesis of modified oligonucleotides is an alluring strategy since a single modified nucleotide could be employed for the construction of numerous oligonucleotides potentially containing multiple modified C-nucleotides irrespective of the length of the sequence. [START_REF] Johnson | Enzymatic Incorporation of a Coumarin-Guanine Base Pair[END_REF][START_REF] Li | Polymerase-Mediated Site-Specific Incorporation of a Synthetic Fluorescent Isomorphic G Surrogate into RNA[END_REF] Our study shows that the C-nucleotide analog dBphTP can be used as a building block for the enzymatic construction of modified oligonucleotides under certain experimental conditions. We have shown that the presence of consecutive modified residues on template oligonucleotides can partially improve the incorporation efficiency of dBphMP into DNA even though the yields remain modest (~50% conversion was obtained with the Dpo4 polymerase). However, the incorporation efficiency can be further improved by changing the stereoelectronic nature of the distal ring of templating dBph nucleotides. Best incorporation efficiency was obtained by introducing a para-amino moiety on the distal ring combined with the Dpo4 or the Kf exo - polymerases. Moreover, multiple dBph building blocks can be incorporated into DNA by the Therminator, Dpo4, or Kf exo - polymerases either by using templates containing abasic sites or by the TdT-tailing reaction which seems to be restricted to only four incorporation events. We have also devised a two-step enzymatic protocol that permits the synthesis of any sequence containing a dBph moiety. This two-step method can potentially be applied to other modified (e.g. pyrene-modified nucleotides [START_REF] Matray | A specific partner for abasic damage in DNA[END_REF]) or natural (e.g. dATP [START_REF] Röthlisberger | Incorporation of a minimal nucleotide into DNA[END_REF]) nucleotides that are known to be incorporated opposite basic sites. Collectively, these results demonstrate the possibility of using polymerases to synthesize dBph-containing oligonucleotides and could be expanded to other C-nucleotides. This approach can benefit from the use of engineered polymerases [START_REF] Pinheiro | Synthetic Genetic Polymers Capable of Heredity and Evolution[END_REF][START_REF] Chen | Evolution of thermophilic DNA polymerases for the recognition and amplification of C2 '-modified DNA[END_REF][START_REF] Marx | The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases[END_REF] and work towards this aim is currently under way in our laboratory.

Experimental section

Materials and methods

All reactions were performed under argon in flame-dried glassware. Anhydrous solvents for reactions were obtained from Sigma Aldrich. Flash chromatography was performed using silica gel (230-400 mesh) from Sigma Aldrich or on a Reveleris Prep system from Büchi. Thin layer chromatography was carried out on pre-coated glass-backed plates of silica gel (0.25 mm, UV 254 ) from Macherey-Nagel. All chemicals and solvents used were purchased from Sigma-Aldrich and Alfa Aesar unless stated otherwise. NMR spectra were recorded on a Bruker Avance 400 spectrometer (400.13 MHz for 1 H, 100.62 MHz for 13 C, and 161.62 MHz for 31 P) and all spectra were referenced to the signals of the corresponding solvent. Chemical shifts are given in ppm (δ scale) and coupling constants (J) in Hz. Assignation of the NMR signals was performed by using a combination of 1 H/ 1 H-COSY, 13 C-DEPT-135, and 13 C/ 1 H-HMBC experiments. High resolution electrospray ionization (ESI) mass spectra (MS, m/z) were recorded on a Waters Q-Tof Micro MS in the positiveion electrospray ionization (ESI+) mode. Solutions were prepared using 1:1 MeCN/H 2 O containing 0.1% formic acid or MeOH/water containing 10 mM ammonium acetate in the case of sensitive compounds. HPLC purification was performed using an Äkta™ pure system (GE Healthcare) equipped with Thermo Scientific™ DNAPac™ PA100 semi preparative ion exchange column (13µm, 250 x 9.0 mm). Unmodified DNA oligonucleotides were purchased from Microsynth. DNA oligonucleotides containing modified nucleotides were synthesized on an H-8 DNA synthesizer from K&A on a 1 µmol scale. Natural DNA phosphoramidites (dT, dC4bz, dG2DMF, dA6Bz) and solid support (dA6Bz-lcaa-CPG 500Å) were all purchased from ChemGenes. Natural DNA phosphoramidites as well as the modified phosphoramidite were prepared as 0.07 M solutions in MeCN and were coupled using 50 sec and 490 sec steps, respectively. 5-(ethylthio)-1H-tetrazole (0.25 M in MeCN) was used as coupling agent. Capping, oxidation, and detritylation were performed using standard conditions. Cleavage from the solid support and deprotection of oligonucleotides was achieved by treatment with concentrated ammonia at 55°C for 16 h. After centrifugation, the supernatants were collected and the resulting solutions were evaporated to dryness on a speed-vac. Crude oligonucleotides were purified by anion exchange HPLC (Dionex -DNAPac PA200). Buffer solutions of 25 mM Tris-HCl in H 2 O, pH 8.0 (buffer A) and 25 mM Tris-HCl, 1.25 M NaCl in H 2 O, pH 8.0 (buffer B) were used. The purified oligonucleotides were then desalted with SepPack C-18 cartridges. Oligonucleotide concentrations were quantitated by UV spectroscopy using a UV5Nano spectrophotometer (Mettler Toledo). The chemical integrity of oligonucleotides was assessed by UPLC-MS analysis: UPLC was performed on a BEH C18 column (130 Å, 1.7 μm, 2.1 mm x 50 mm) from Waters, installed on an ACQUITY UPLC H-Class System (SQ Detector 2). A Buffer containing 20 mM TEA and 400 mM HFIP in H 2 O was used with a linear gradient from 18 to 31% Methanol within 5 minutes and a flow rate of 0.3 mL/min. All the DNA polymerases (Phusion, Hemo KlenTaq, Taq, Bst, Q5, Therminator, Vent (exo -), Dpo4, Deep Vent, Phi29, and Kf exo -) were purchased from New England Biolabs as well as the natural dNTPs. Acrylamide/bisacrylamide (29:1, 40%) was obtained from Fisher Scientific. Visualization of PAGE gels was performed by fluorescence imaging using a using a Storm 860 or a Typhoon Trio phosphorimager with the ImageQuant software (both from GE Healthcare).

Chemical synthesis of nucleotides

Synthesis of nucleoside analog 3:

The starting material 2 [START_REF] Brotschi | Bipyridyl-and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking[END_REF][START_REF] Brotschi | DNA with hydrophobic base substitutes: A stable, zipperlike recognition motif based on interstrand-stacking interactions[END_REF] (100mg, 017mmol) was dissolved in dry pyridine (2.5 mL) at RT under N 2 and DMAP (24 mg, 019 mmol, 1.1eq.) and Et 3 N (61 µL, 0.43 mmol, 2.5 eq.) was added. To this solution acetic anhydride (24 µL, 0.26mmol, 1.5 eq.) was added in one portion. The reaction mixture was stirred for 2.5h then concentrated to dryness. The crude oil was extracted in DCM (3 x 10mL) and NaHCO 3 sat (5 mL). The organic layers were combined dried over MgSO 4 and concentrated. The crude product was purified by flash chromatography (Hex/EtOAc 4/1) to yield 102 mg (95%) of the title compound 3.

R f (Hex

Synthesis of nucleoside analog 4:

Nucleoside 3 (90mg, 0.14 mmol) was dissolved in dry DCM (9 mL) and then treated with dichloroacetic acid (120µL 1.4mmol, 10eq.). The resulting orange reaction mixture was stirred for 30 min. under N 2 at RT. The reaction mixture was quenched with MeOH and evaporated to dryness. The crude oil was extracted in DCM (3 x 10mL) and NaHCO 3 sat (5 mL). The organic layers were combined dried over MgSO 4 and concentrated. The crude product was purified by flash chromatography (Hex/EtOAc 4/1) to yield 37 mg (81%) of the title compound 4.

R f (Hex

Synthesis of nucleoside triphosphate 1:

The suitably protected nucleoside 4 (37 mg, 0.11 mmol) was dissolved in dry pyridine (0.2 mL) and dry dioxane (0.4 mL) at RT under N 2 . To this clear solution, 2-chloro-1,3,2benzodioxaphosphorin-4-one (40 mg, 0.18 mmol, 1.5 eq.) were added and the reaction mixture was stirred for 45 min. A solution of tributylammonium pyrophosphate (95 mg, 0.17 mmol, 1.4 eq.) in dry DMF (0.17 mL) and tributylamine (60 μL) was added dropwise and the reaction mixture stirred for another 45 min. The reaction mixture was then oxidized by the addition of iodine (56 mg, 0.21 mmol, 1.8 eq.) in pyridine (0.98 mL) and H 2 O (0.02 mL). After 30 min of stirring the excess of iodine was quenched with a sodium thiosulfate (10% w/v in water) and the clear solution was concentrated under reduced pressure at 30°C. The concentrated mixture was treated with ammonium hydroxide 30% (12 mL) for 2h. The yellow suspension was again concentrated under reduced pressure at 30°C. The yellow residue was dissolved in H 2 O (2 mL) and precipitated by the addition of NaClO 4 2% in acetone (12 mL). The crude product was purified by RP-HPLC (30% B in 20 min; Buffer A) TEAB 50mM pH=8; Buffer B) TEAB 50mM pH=8, 50% ACN) to yield 29 mg (48 %) of the pure triphosphate 1.

MS (MALDI

Enzymatic reactions

The 5'-FAM-labelled primer P1 (30 pmol) was annealed to the appropriate template (45 pmol) in water by heating to 95°C and then gradually cooling to room temperature. The appropriate reaction buffer, the modified triphosphate; and DNA polymerases were added to the annealed oligonucleotides for a total reaction volume of 20 µL. Following incubation at the optimal temperature for the enzyme, the reactions were quenched by adding stop solution (20 µL; formamide (70%), ethylenediaminetetraacetic acid (EDTA; 50 mm), bromophenol (0.1%), xylene cyanol (0.1 %)). The reaction mixtures were subjected to gel electrophoresis in denaturing polyacrylamide gel (20%) containing trisborate-EDTA (TBE) 1x buffer (pH 8) and urea (7M). Visualization was performed by phosphorimaging.

For TdT-tailing reactions, solutions containing 20 or 2 pmol primer P1, 1 U μL -1 of TdT, dBphTP (200 μM), 1× reaction buffer, and H 2 O (for a total reaction volume of 10 μL) was incubated at 37 °C for 5 h. The reaction mixtures were then purified by Nucleospin columns and quenched by the addition of an equal volume of loading buffer. The reaction products were then resolved by electrophoresis (PAGE 20 %) and visualized by phosphorimager analysis. Fig. S3. Gel analysis (PAGE 20%) of products stemming from various bypass experiments. PEX reactions with natural controls were carried out using template T14, primer P1, and incubate at the adequate temperature for 1 h. The modified dBph unit was incorporated opposite a single abasic site (template T9 as described in Fig. S2) and natural dNTPs were then added and incubated for an additional hour. P indicates the unreacted primer. Polymerases used: 1. Bst; 2. Therminator; 3. Dpo4; 4. Kf exo -. Table S1. Yields (in %) of n+1 product formation based on the gel analysis shown in Figures 4 and5.

Polymerase

NMR and MALDI spectra

Fig. 1 H NMR spectrum of compound 3.

Fig. S5. 13 C NMR spectrum of compound 3.

Oligonucleotide synthesis and characterization:

Table S2. Overview of oligonucleotides containing biphenyl and modified biphenyl nucleotides.

Template

Figure 2 .

 2 Figure 2. Chemical structures of the modified biphenyl-C-nucleoside (5-10) and abasic site nucleoside phosphoramidite 11 used for the synthesis of modified oligonucleotides.

Name Sequence (shown in the 5 '

 5 to 3' direction) a P1 5′-TAC GAC TCA CTA TAG CCT C T1 5′-GGA GBphG AGG CTA TAG TGA GTC GTA T2 5′-GGBph BphBphG AGG CTA TAG TGA GTC GTA T3 5′-GGA GXG AGG CTA TAG TGA GTC GTA T3 5′-GGA G5G AGG CTA TAG TGA GTC GTA T4 5′-GGA G6G AGG CTA TAG TGA GTC GTA T5 5′-GGA G7G AGG CTA TAG TGA GTC GTA T6 5′-GGA G8G AGG CTA TAG TGA GTC GTA T7 5′-GGA G9G AGG CTA TAG TGA GTC GTA T8 5′-GGA G10G AGG CTA TAG TGA GTC GTA T9 5′-GGA GG AGG CTA TAG TGA GTC GTA T10 5′-GGG AGG CTA TAG TGA GTC GTA T11 5′-G AGG CTA TAG TGA GTC GTA T12 5′-GGA GCG AGG CTA TAG TGA GTC GTA a Numbers ranging from 5 to 10 represent the modified nucleotides shown in Figure 2 and  = 11.

Figure 3 .

 3 Figure 3. Gel analysis (PAGE 20%) of the products from primer extension reactions with primer P1, template T2, and the modified nucleotides dTP and dBphTP. All the reaction mixtures contained 200 µM of nucleotides and 2 U of the DNA polymerases. The reaction mixtures were incubated at the corresponding temperatures in 1X reaction buffer for 3 h. P indicates the unreacted primer. Polymerases used: 1. Phusion; 2. Hemo KlenTaq; 3. Q5 DNA polymerase; 4. Bst; 5. Taq; 6. Therminator; 7. Vent (exo -); 8. Dpo4; 9. Deep Vent; 10. Phi29; 11. Kf exo -.

Figure 4 .

 4 Figure 4. Gel analysis (PAGE 20%) of the products of PEX reactions with dBphTP, primer P1, and templates A) T3, B) T5, and C) T6. All the reaction mixtures contained 200 µM of nucleotides and 2 U of the DNA polymerases. The reaction mixtures were incubated at the corresponding temperatures in 1X reaction buffer for 3 h. P indicates the unreacted primer. Polymerases used: 1. Phusion; 2. Hemo KlenTaq; 3. Q5 DNA polymerase; 4. Taq; 5. Bst; 6. Therminator; 7. Vent (exo -); 8. Dpo4; 9. Deep Vent; 10. Phi29; 11. Kf exo -.

Figure 5 .

 5 Figure 5. Gel analysis (PAGE 20%) of the products of PEX reactions with dBphTP, primer P1, and templates A) T7, B) T4, and C) T8. All the reaction mixtures contained 200 µM of nucleotides and 2 U of the DNA polymerases. The reaction mixtures were incubated at the corresponding temperatures in 1X reaction buffer for 3 h. P indicates the unreacted primer. Polymerases used: 1. Phusion; 2. Hemo KlenTaq; 3. Q5 DNA polymerase; 4. Taq; 5. Bst; 6. Therminator; 7. Vent (exo -); 8. Dpo4; 9. Deep Vent; 10. Phi29; 11. Kf exo -.

Figure 6 .

 6 Figure 6. Gel analysis (PAGE 20%) of the products of PEX reactions with dBphTP, primer P1, and templates A) T10 and B) T11 under various experimental conditions. All the reaction mixtures contained 2 U of the DNA polymerases and the reaction mixtures were incubated at the corresponding temperatures in 1X reaction buffer for 3 h. The final concentration of Mn 2+ was 1 mM. P indicates the unreacted primer. Polymerases used: 1. Bst; 2. Therminator; 3. Dpo4; 4. Kf exo -.

Figure 7 .

 7 Figure 7. Gel image (PAGE 20%) of the TdT-mediated tailing reactions with dBphTP and different initiator concentrations, reaction times, and cofactors. All reactions were performed with primer P1 and incubated with 10 U of TdT at 37°C. Cofactor concentrations were 1 mM for Mn 2+ and Mg 2+ and 0.25 mM for Co 2+ . P indicates the unreacted primer.

Fig. S2 .

 S2 Fig. S2. Gel analysis (PAGE 20%) of PEX reactions with primer P1, template T9 A), T10 B) , and T11 C) in the presence of dBphTP. All the reaction mixtures contained 200 µM of nucleotides and 2 U of the DNA polymerases. The reaction mixtures were incubated at the corresponding temperatures in 1X reaction buffer for 1 h. P indicates the unreacted primer. Polymerases used: 1. Phusion; 2. Hemo KlenTaq; 3. Q5 DNA polymerase; 4. Bst; 5. Taq; 6. Therminator; 7. Vent (exo -); 8. Dpo4; 9. Deep Vent; 10. Phi29; 11. Kf exo -.

Fig. S6. 1 H

 1 Fig. S6. 1 H NMR spectrum of compound 4.

Fig. 13 C

 13 Fig.13 C NMR spectrum of compound 4.

Fig. S8. 1 H

 1 Fig. S8. 1 H NMR spectrum 1.

Fig. S9. 31 P

 31 Fig. S9. 31 P NMR spectrum of 1.

Fig. S10 .

 S10 Fig. S10. MALDI MS spectrum of dBphTP 1.

= 5 ;

 5 X2 = 6; X3 = 7; X4 = 8; X5 = 9; X6 = 10. b data taken from Tet. Lett. 2018, 59, 4241-4244.

Fig. S11 .

 S11 Fig. S11. Total ion chromatogram (TIC) of template T1.

Fig. S12 .

 S12 Fig. S12. Mass spectrum of template T1.

Fig. S13 .

 S13 Fig. S13. Total ion chromatogram (TIC) of template T2.

Fig. S14 .

 S14 Fig. S14. Mass spectrum of template T2.

Fig. S15 .

 S15 Fig. S15. Total ion chromatogram (TIC) of template T3.

Fig. S16 .

 S16 Fig. S16. Mass spectrum of template T3.

Fig. S17 .

 S17 Fig. S17. Total ion chromatogram (TIC) of template T4.

Fig. S18 .

 S18 Fig. S18. Mass spectrum of template T4.

Fig. S19 .

 S19 Fig. S19. Total ion chromatogram (TIC) of template T5.

Fig. S20 .

 S20 Fig. S20. Mass spectrum of template T5.

Fig. S21 .

 S21 Fig. S21. Total ion chromatogram (TIC) of template T6.

Fig. S22 .

 S22 Fig. S22. Mass spectrum of template T6.

Fig. S23 .

 S23 Fig. S23. Total ion chromatogram (TIC) of template T7.

Fig. S25 .

 S25 Fig. S25. Total ion chromatogram (TIC) of template T8.

Fig. S26 .

 S26 Fig. S26. Mass spectrum of template T8.

Table 1 .

 1 Primers and templates used for primer extension reactions.

  / EtOAc 4/1) = 0.4. MS C 40 H 38 O 6 Na + calculated 637.256; observed 637.2585.

	1 H NMR (400 MHz, Chloroform-d) δ = 7.61 (m, 4H), 7.52
	(dd, J=7.8, 2.1, 4H), 7.46 (m, 2H), 7.43 -7.36 (m, 4H), 7.33 -
	7.27 (m, 2H), 7.26 -7.22 (m, 1H), 6.94 -6.65 (m, 4H), 5.44 -
	5.31 (m, 1H), 5.18 (dd, J=10.9, 5.0, 1H), 4.23 (d, J=2.0, 1H),
	3.97 -3.74 (m, 6H), 3.47 -3.29 (m, 2H), 2.46 -2.34 (m, 1H),
	2.25 (ddd, J=13.6, 11.0, 5.9, 1H), 2.13 (s, 3H).
	13 C NMR (101 MHz, Chloroform-d) δ = 170.5, 158.5, 144.9,
	140.9, 140.7, 140.1, 136.1, 136.1, 130.2, 128.8, 128.3, 127.8,
	127.3, 127.2, 127.1, 126.8, 126.6, 113.1, 86.3, 84.3, 80.2, 64.3,
	55.2, 41.6, 21.2.

  / EtOAc 4/1) = 0.15. MS C 19 H 20 O 4 Na + calculated 312.1362; observed 637.2585.

	1 H NMR (400 MHz, Methanol-d 4 ) δ = 7.65 -7.56 (m, 4H),
	7.54 -7.48 (m, 2H), 7.47 -7.40 (m, 2H), 7.35 -7.30 (m, 1H),
	5.28 (ddd, J=6.0, 1.8, 0.9, 1H), 5.12 (dd, J=11.0, 5.0, 1H), 4.08
	(td, J=4.4, 2.2, 1H), 3.79 (dd, J=4.5, 1.2, 2H), 3.33 (p, J=1.7,
	1H), 2.35 (ddd, J=13.7, 5.0, 1.1, 1H), 2.12 (s, 3H), 2.15 -2.06
	(m, 1H).
	13 C NMR (101 MHz, Methanol-d 4 ) δ = 171.0, 140.8, 140.7,
	140.0, 128.4, 127.0, 126.6, 126.5, 126.4, 85.7, 80.2, 76.8, 62.3,
	41.1, 19.6.

  -): m/z calculated for C 17 H 19 O 12 P 3 P NMR (162 MHz, Deuterium Oxide) δ = -6.70 (d, J=13.8), -9.78 (d, J=19.1), -21.28 (t, J= 14.6).

	2-: 508.01;
	observed: 508.24.
	1 H NMR (400 MHz, Deuterium Oxide) δ = 7.72 -7.57 (m,
	4H), 7.50 (d, J=8.0, 2H), 7.43 (t, J=7.7, 2H), 7.38 -7.31 (m, 1H),
	5.16 (dd, J=10.5, 5.7, 1H), 4.52 (d, J=5.4, 1H), 4.14 (s, 1H), 4.06
	(t, J=4.8, 2H), 2.26 -2.08 (m, 2H).

31

Acknowledgments

The authors gratefully acknowledge financial support from Institut Pasteur. The authors thank Dr. Luke McKenzie for the recoding of the MALDI spectrum of the modified triphosphate and Ms. Marie Flamme for some gel images.

Supplementary Material

Supplementary data (additional gel images, NMR and LC-MS characterization of modified oligonucleotides) can be found associated with this article.

SUPPORTING INFORMATION

Enzymatic synthesis of biphenyl-DNA oligonucleotides


Damien Sileo

Camille Pradel

Guillermo Echegoyen

Anselmo Peña

Arantxa Otegi

Jan Milan Deriu

Mark Cieliebak

Ander Barrena

Eneko Agirre

Camille Prade1

Guillermo Eschegoyen

Anselmo Pef1as

Matching Words and Knowledge Graph Entities with Meta-Embeddings

Keywords: Question answering, Knowledge Graphs, Word Embeddings, Graph Embeddings, Meta Embedding

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Structured data has become ubiquitous, abundant and involved in numerous applications. Knowledge bases like DBpedia, Wikidata, Op n ye [START_REF] El Farber | A comparative sur vey of dbpedia., freebase, opencyc, wiki data[END_REF] provide large and growing structured resources. They contain millions of facts represented as triplets such as (Paris, LOCATED_JN, France). Form al languages such as SPARQL and scalable endpoint architecture allow effi cient queries. However, natural language is more conve nient for mo t user . 'fianslating natural language que ries into formal language queries (e.g. SPARQL) has been a Jong tanding artificial intelligence task. Table 1 shows an example of a natural language question with associated SPARQL query. But current systems are only successful on restricted versions of this task, e.g. u ing specific patterns [PHH13, T 1DL17].

ince full translation-based sy terns are not re li able, a useful task would be the matching of related SPARQL requests (either from historical data or from the output of a translation-based y tern) according to their imilarity to a natural language question. In this pap er, we tackle the prediction of imilarity between natural language questions and PARQL requests. Word embeddings are a key component in many tex tual similarity systems and have been used to represent natural language questions. However the comp onents of SPARQL queries are either SPARQL keywords (e.g. SELECT) or Uniform R. essource Unifiers (UR.I) (e.g. http :/ /dbpedia.org/resource/Stanley_l{ubrick ).

There exists pre-computed URI embeddings, but learning an alignment of the embeddings latent space is needed for similarity computations and relying on task pecific manually annotated data is co tly. Meta embeddings could be used in order to olve this pro blem. A metarembedding is a repre entation derived from a set of distinct embeddings (e.g. Word2Vec and GloVe). Yet there exists no meta-embedding me thod leveraging pretrained knowledge graph embed dings and word embeddings. Such meta embedding could also allow integration of symbolic external know ledge for common sense reasoning or information retrie val.

Our contributions are as follow :

-A meta-embedding method to align word embed dings with graph entities embeddings Pair Score

Feed-Forward RDF2Vec inherits many properties from Word2Vec vectors (e.g. a co ine imilarity that reflect related ness).

In this work, we will compare the use of RDF2Vec and Word2Vec for URI representation, and propose a meta-embedding method to combine them.

Similarity estimation models

A po sible use-case of our method is improvement on similarity prediction between a natural language quetion and a SPARQL query. In thi work, we use a sia mese neural network whose architecture i depicted in the figure 1 for uch similarity estimation in a super vised setup. We experimented using tate of the art [BSAl ] models but they did not perform well proba bly due to the hort contexts of querie as opposed to the evaluation datasets used in entity linking literature.

We represent the que tions/queries with the ave rage of its ymbols (words/URI) embeddings, com po ed with a matching function (with a concatena tion of hadamard product, abs olute difference of in put vectors) foll owed by a feed-forward neural network. Average-pooling is a implistic sequence encoding me thod but it was shown to be competitive with more

complicated architectures [sww + 1 ] .
Representing word from natural language questions is straightforward using word embeddings. By contrast, there are several wa y s to represent DBPedia URI (e.g. http : // dbpedia.org/resource/Stanley J<ubrick ) . For instance text can be derived from the label for the URI (e.g Stanley Kubrick) allowing the use of word Word and graph embedding encode complementary knowledge, but their latent pace need to be aligned in order to perform similarity computations. Here, we propo e to learn to map the latent space of RDF2Vec to the space of word embeddings.

To do so, we train a feed-forward neural network J 9 in order to predict the word embedding Word2Vec(u) representation of a given URI u from its URI embed ding RDF2Vec(• u). fore pecifically, we optimize 0 in the following loss function : A fo(RDF2Vec(u)) is trained to lie in the same space as Word2Vec(u), a weighted average of these re pre entations can be also used :

L = L MSE(Word2Vec(u), fo(RDF2Vec(u))) (1)
Weighted0 (u) = (1-o-)Word2Vec (u)+o-fo(RDF2Vec(u)) (2)

Experiments

Query matching evaluation

We evaluate our model on the LC-QuAD [TMDLl 7] dataset which is a col lection of 5000 natural language questions with associated SPARQL querie . 4000 pair are used for training and the remaining is used for eva luation . For each example, we generate 9 example of dissimilar L/SPARQL queries using random as ocia tions of different querie . This proces is done on train data and test data eparately.

To represent natural language questions, we al ways use word embeddings from [MGB + 1 ] trained on Com monCrawl.

Regarding URI representations we evaluate several embeddings : gw(RDF2Vec) is a linear projection of RDF2Vec em bedding. The projection W is initialized randomly and learnt during the matching prediction training.

Jo is instanciated with a two hidden layer MLP (hid den layer size are 200,200) with batch-normalization and ReLu activation. 0 is trained on 6.0M URIS, using 1 epoch and using Adam optimizer [START_REF] Diederik | Adam : A method for stochastic optimi zation[END_REF] with de fault parameters, using the loss from equation 1. The pararameters are kept fixed in the matching prediction training.

For the matching detection training, 10% of training data is kept aside as validation set in order to determine the best number of epochs (found to be also using Adam optimizer).

We performed cro validation on the parameter a. Figure 3 hows the influence on a, on evaluation me trics. a, = 0 is the ame as only using Word2Vec, and a,= 1 is equivalent to only using fo(RDF2Vec).

Query matching results

Table 2 shows the te t re ults of different methods. Since accuracies are high, we also report cro s entropy for a more meaningful comparison. U ing the word em beddings of label already yields high re ults. However, when combined with aligned graph embedding with the Weighted method the result are significantly bet ter.

Intrinsic Evaluation

We also evaluate our meta-embedding intrinsically with a standard word similarity prediction evaluation : we use word embeddings to predict cosine similarity between word pairs, and measure the pear son correla tion between cosine similarity and human judgments from imilarity /relatedness prediction datasets. Sim- TABLE 3 -Pearson correlation b tween cosine simi larity of embeddings and human judgmen for eve ral models. We used the best values of o when repor ting the score of Weighted models. CO C is a meta ensembling baseline (concatenation of emebeddings).

Lex [START_REF] Hill | Ko rhonen. Simlex-99 9 : Evaluating seman tic models with genuine imila[END_REF] is a similarity judgement dataset (anto nyms should have a low rating) while ME [START_REF] Bruni | Multimodal distributional seman tics[END_REF] and M'Turk [RAGMll] are relatedness dataset (anto nym can have a high rating).

Once again, we use the Weighted meta-embedding model from equation 2. We report the improvement over the Word2Vec baseline according the the value of of o. Figure 5 show the re ul over v a rious datasets.

We also performed the same experiment using Word-et2Vec [BAK + 17] instead of RDF2Vec. Word et2Vec is a graph embedding computed using the Wordnet graph consisting 2 5k relations between words, such as (furniture, is_a, piece_oLfurni ture) \Ale used th same exp rimental etup but perfor med 2 epochs when optimizing £,. The results of best Weighted models are reported in table ??. Our meta embeddings are competitive with CO C while having lower dimensionality (300 vs 1150).

Related Work

Several models exist for meta-embedding [START_REF] Yin | Lear ning word meta-embeddings[END_REF] [ iISLl 7]. However, they use a set of embeddings and a return a meta-embedding lying in a new latent space, except [CBI ] who shows that meta-embedding can be obtained by simply averaging or concatenating a et of input embeddings.

Retrofitting models [FDJ + 15, ] also improve embed ding by leveraging knowledge graphs, in a different way : they use pre-computed word embeddings and tune word representations so that they fulfill some constraints dictated by the knowledge graph.

The most similar approach to our is [START_REF] Mikolov | Exploiting similarities among languages for machine translation[END_REF] where embeddings in different language ( e.g. french and en glish) are aligned using a translation matrix learn on a limited size multilingual lexicon.

The pecificity of our best model is that it is additive. With proper cro s validation, the weighted version of our method can ensure better or equal re ults.

Another line of work deal with Alignment of know ! dg from textual data and graph data. It has be n explored with joint learning of emb ddings from lan guage model and knowledge graph link prediction [ABMiKl ] . However, tho method are less flexible, and ca not leverage the high quality word embed dings trained on mas ive textual datasets without a re-training from scratch.

Conclusion

We proposed a simple, flexible meta.-embedding me thod based on word embeddings and labelled graph embedding and reported significant improvement on word representation and SPARQL queries/natural lan guage matching. It can be applied to other graphs such as UMLS [BKF + l ] for biomedical LP or social net works graphs [START_REF] Leskovec | S AP Dataset : tanford large network dataset collection[END_REF]. Other languages can be used as well. We expect more sub tantial gains on low resource language where corpus sizes a.re more limited.

FIGURE 1 -

 1 FIGURE 1 -Model architecture for similarity estima tion

  uEVV is the et of training examples, i.e. the et of URis where a word in a label matches a word vector. When several words are found, we use the average of their embedding . Figure2illustrates this approach.

FIGURE 2 -

 2 FIGURE 2 -Model architecture for embedding alignment FIGURE 3 -Influence of a, on matching prediction va lidation results

FIGURE 4 -

 4 FIGURE 4 -Influence of o on word similarity pre diction evaluation using the weighted combinati on of Word2Vec and fo(RDF2Vec). y axis is the pearson cor relation improvement over the v\Tord2Vec baseline.

FIGURE 5 -

 5 FIGURE 5 -Influence of o on word similarity pre diction evaluation using the weighted combination of Word2Vec and fo(WordNet2Vec). y axi is the pear on correlation improvement over the Word2Vec baseline.

TABLE 2 -

 2 //dbp dia.org/ontology/director> <http ://dbpedia.org/r ource/Stanley..Kubric k> Test results of different URI embeddings models; bold value denotes the best results Weighted is defined in equation 2

	1. (2016-04 version)

½ (Word2Vec(Stan/ey) + Word2Vec(Kubrick))

; -.,.,

Acknowledgments

Thi work has been upported by ERA-et CHIST ERA LIHLITH Project funded by the Agencia Es tatal de Investigaci6n (AEI, pain} projects PCIN-2017-11 /AEI and P -2017-0 5/AEI the Agence a.tionale pour la Recherche (A R, France) pr ojects R-17-I-IR2-0001-03 and R-17-CI-IR2-0001-04, and the Swiss a.tiona.l cience Foundation (S F, Swit zerland) project 20Cl-l21 174237.


Fabienne Levi-Acobas

Adam Katolik

Pascal Röthlisberger

Thomas Cokelaer

Ivo Sarac

Masad J Damha

Christian J Leumann

Marcel Hollenstein
email: marcel.hollenstein@pasteur.fr

Compatibility of 5-ethynyl-2'F-ANA UTP with in vitro selection for the generation of base-modified, nuclease resistant aptamers

Xeno nucleic acids (XNAs) are analogs of the two natural genetic polymers (DNA and RNA) bearing modifications at the level of the sugar. [1][2][3][4] The presence of these chemical modifications provides XNAs with properties that are absent or muted in corresponding natural systems. For instance, the presence of sugar modifications markedly improves the nuclease resistance of oligonucleotides which is of highly relevant for therapeutic [5][6][7][8][9] and in vivo applications. 10, 11 Similarly, base-modifications convey orthogonality to modified nucleic acids [12][13][14][15][16][17][18][19] and provide functionalities to synthetic genetic polymers, which improve their catalytic [20][21][22] and/or binding capacities. [23][24][25] The synthesis of XNAs proceeds either via a chemical route (using phosphoramidites during automated solid-phase synthesis) or a chemoenzymatic method, whereby a polymerase incorporates modified nucleotides. 26, 27 The latter method endows some advantages compared to solid phase synthesis, including: 1) high functional group tolerance; 2) unrestricted oligonucleotide length; and 3) compatibility with SELEX and related combinatorial in vitro selection methods. Combined with recent progress in polymerase engineering 28 these intrinsic properties have instigated the selection of numerous XNA aptamers [29][30][31][32][33][34][35][36][37] and catalysts. [38][39][40] Synthesis of the modified nucleotide started with the bromination of the anomeric center of the commercially available nucleoside 1 (Scheme 1). The presence of the 2'fluorine ensures that the bromine atom is installed in the α configuration in compound 2. The expected South pucker of arabinose places the halogens at C1' and C2' in pseudoaxial positions, which are stabilized by gauche (O4'-C1'-C2'-F2') and anomeric effects (O4'-C1'-Br), respectively.

2 | J. Name., 2012, 00, 1-3
Please do not adjust margins Please do not adjust margins nucleobase was then installed on the sugar moiety by application of modified Vorbrüggen conditions. 55, 56 This reaction is always in competition with the analogous S N 1 reaction, which forms a mixture of both anomers. The contribution of S N 1 and S N 2 depends mostly on the solvent and the temperature, and generally the combination of low solvent polarity and elevated temperature renders acceptable results. We therefore chose toluene and 80°C overnight reflux as reaction conditions (see Supporting Information) and both anomers were formed (1:10 α/β), which could be separated by chromatography yielding compound 3 in moderate yields. Removal of the blocking groups of 3 was followed by 5'-DMTr protection yielding 5 in good yields (57% over two steps). Acetylation and detritylation of nucleoside 5 led to the suitably 3'-O-protected nucleoside 7, which was subjected to Ludwig-Eckstein conditions 57 to generate the desired 5'-triphosphate 8. The low yields obtained during this last step stem from a rather lengthy purification protocol and highlight the need for more robust and generally applicable protocols for the synthesis of nucleoside triphosphate analogs. With 5-ethynyl-2'F-ANA UTP 8 at hand, we proceeded to evaluate the capacity of polymerases to incorporate this modified nucleotide into DNA. To do so, primer extension (PEX) reactions were carried out with various polymerases (Therminator, Vent (exo -), Bst, Deep Vent, the Klenow fragment of DNA polymerase I exo -(Kf exo -), and Taq) and by using triphosphate 8 instead of its natural counterpart dTTP. The possibility of incorporating nucleotide 8 was first evaluated using the 37-nucleotide long template T1 and the 5'-FAM-labelled, 21-mer primer P1 (see Table S1 for the sequence compositions). With the exception of Bst, all polymerases led to the full conversion of primers to the expected full-length products with minimal truncated sequences (Figure S1). Next, we evaluated the possibility of incorporating multiple, consecutive modifications. To this end, we applied template T2, which contains seven adenine nucleotides located immediately 5'-downstream to the 3'-end of primer P2. 59 In this context, Vent (exo -) polymerase managed to incorporate six modified nucleotides while Therminator produced full length products accompanied by some non-templated addition (Figure S2). 60 Moreover, Deep Vent, Taq, and Kf exo -also incorporated the modified nucleotide albeit less efficiently, while Bst did not incorporate any modified nucleotide. A similar preference for family B over family A polymerases was observed previously with unmodified 2'F-ANA-nucleotides, suggesting that the ethynyl moiety has a negligible impact on the incorporation efficiency. 47, 48 Surprisingly, nucleotide 8 is a rather poor substrate for polymerases under PCR conditions since only Therminator led to amplified full-length products albeit in lower yields compared to the control reaction performed with natural dNTPs (Figure S4). Having identified conditions, which enable the polymerization of the modified nucleotide, we wanted to ascertain that the ethynyl moiety, once installed in 2'F-ANA-modified oligonucleotides, the ethynyl moiety was still accessible to further chemical modification by the CuAAC reaction. Thus, PEX reactions were carried out with the T3/P3 system. The resulting products were then incubated with an azide-modified biotin derivative (obtained by amide coupling of biotin with 2azidoethanamine 61

) under click reaction conditions (see Supporting Information). Next, the biotin-modified, 5-ethynyl-2'F-ANA -containing oligonucleotides were incubated with avidin and analyzed using gel electrophoresis. 42 Figure 1 clearly shows that biotinylated avidin treated 2'F-ANAoligonucleotides from Therminator PEX reactions are unable to enter the gel due to the presence of the protein (lane 8). A similar trend can be observed when using Vent (exo -) as a polymerase, albeit in lower yields (lane 7). Lastly, to show applicability for SELEX experiments, it was necessary to demonstrate that: 1) modified DNA synthesis did not encounter misincorporation events; and 2) sequences Please do not adjust margins Please do not adjust margins containing 5-ethynyl-2'F-ANA-U modifications could be reverse-transcribed back to unmodified DNA. To do so, first, the PEX reactions proceeded with a 98-nucleotide long template T4 and a 25-mer primer P4 (Figure S3). 60 Next, exonuclease was applied to digest and remove the unmodified template strand and the modified strand served as a template for DNA synthesis under PCR conditions (Figure 2A). Of the different polymerases tested, Q5 DNA polymerase led to strong amplification of the modified template. The resulting dsDNA product was then introduced into a library, which was subjected to Illumina sequencing (see Supporting Information). Data processing involved phix removal, mapping the data on the sense strand, and analysing the concordance (Figure 2B) as well as the number of errors per read (Figure S4). This analysis showed that 99.2% of the reads have a concordance > 95% and that 95% of all reads have 2 or less errors with no detectable single nucleotide variants (SNVs) in any of the reads. 62 Lastly, the very low rate of base substitution (<0.5%) further confirms the high fidelity of the DNA-modified DNA-DNA cycle.

Conclusions

In this report, we produced an XZA nucleoside triphosphate bearing two modifications consisting of a 2'F-ANA-sugar moiety and a C5-modified nucleobase. We also highlighted its compatibility with enzymatic synthesis of modified DNA and the possibility of using the ethynyl anchor for the introduction of additional functional groups. Lastly, sequencing and data analysis revealed the fidelity of incorporating the modified nucleotide into DNA and the efficiency of the conversion of 2'F-ANA-containing oligonucleotides back to DNA. The efficiency of 5-ethynyl-2'F-ANA-synthesis might be further improved by using engineered polymerases 31 and the sequencing protocol simplified by application of nanopore sequencing. 63 Work towards these aims as well as application of the click-SELEX protocol 24, 64 to identify aptamers featuring high binding affinity and nuclease resistance is currently ongoing.

58 Scheme 1 .

 581 Scheme 1. Synthesis of 5-ethynyl-2'F-ANA UTP 8. Reagents and conditions: i) HBr/AcOH, 0°C to rt, 12 h; ii) 5-(2-trimethylsilylethynyl)-uracil, BSA, toluene, 80°C, 12h, 42% (over 2 steps); iii) a) NaOH in THF/MeOH/H2O (5:4:1), 0°C, 1h; b) NH4Cl, rt, 20 min, 69%; iv) DMTrCl, pyridine, rt, 12 h, 83%; v) Ac2O, pyridine, 0°C to rt, 1h, 80%; vi) TFA, CH2Cl2, 0°C to rt, 30 min, 45%; vii) a) 2-chloro-1,3,2benzodioxaphosphorin-4-one, pyridine, dioxane, rt, 45 min; b) (nBu3NH)2H2P2O7, DMF, nBu3N, rt, 45 min; c) I2, pyridine, H2O, rt, 30 min; d) NH3(aq.), rt, 1.5 h, 2% (4 steps).

Figure 1 .lanes 4 :

 14 Figure 1. Gel analysis (PAGE 20%) of the functionalization of 2ˈF-ANAoligonucleotides by the CuAAC reaction. PEX reactions were carried out with the 31-mer long template T3 and the 15-mer primer P3 leading to oligonucleotides containing four 5-ethynyl-2'F-ANA nucleotides. Lanes 1: PEX reactions with natural dNTPs and Taq polymerase; lanes 2: T4 DNA polymerase; lanes 3: Kf exo -; lanes 4: Bst; lanes 5: Taq polymerase; lanes 6: Deep Vent; lanes 7: Vent (exo -); lanes 8: Therminator.

Figure 2 .

 2 Figure 2. A) Gel analysis (agarose 2%) of reverse-transcription reactions using a 98 nucleotide long FANA-modified template and natural dNTPs. Lane 1: Phusion DNA polymerase and modified template; lane 2: Q5 DNA polymerase and modified template; lane 3: KOD XL DNA polymerase and modified template; lane 4: Taq DNA polymerase and unmodified DNA template; lane 5: Taq DNA polymerase, unmodified DNA template, and only three natural dNTPs (dATP, dCTP, dGTP). L represents a DNA weight ladder. B) Concordance of all the reads mapped to the Q5 DNA polymerase; concordance is defined as 1 -(D+I+M) / (D+M+I+S) where D, I, S and M stands for deletion, insertion, substitution and match, respectively)

J. Name., 2013, 00, 1-3 | 3

Acknowledgements

We thank Cédric Fund (Biomics Platform, C2RT, Institut Pasteur, Paris, France) for the Illumina sequencing. This work was financially supported by Institut Pasteur (starting grant to M.H.), France Génomique (ANR-10-INBS-09-09) and IBISA (to T.C.), the Natural Science and Engineering Council of Canada (Discovery grant to MJD), and the Swiss National Science Foundation (200020-146646 to C.J.L.).

Conflicts of interest

There are no conflicts to declare.