C Petru

Dandan Su

Maëva Coste

Andrei Diaconu

Mihail Barboiu
email: mihail-dumitru.barboiu@umontpellier.fr

Sébastien Ulrich
email: sebastien.ulrich@enscm.fr

Cationic Dynamic Covalent Polymers for Gene Transfection

Dynamic covalent polymers are materials formed by reversible covalent bonds and non-covalent interactions through an adaptive constitutional dynamic chemistry. The implementation of dynamic covalent polymers in gene delivery has recently emerged due to its responsive and adaptive features. Indeed, such an approach offers the alluring promise of discovering optimal delivery vectors self-fitted to their nucleic acid cargos and responsive to environmental changes (e.g. pH changes, presence of a biomolecular target). This review will discuss more precisely the structural features of the molecular building blocks used so far, the architecture of the resulting dynamic covalent polymers from linear to 2D and 3D, and the covalent and supramolecular self-assembly processes at play in the nucleic acid recognition and delivery, showcasing in particular the very few examples of adaptive self-assembly of dynamic covalent polymers templated by nucleic acids and responsive to the presence of biomolecular targets found in cell membranes which facilitate cell entry.

Introduction

Genetic disorders caused by mutations of genes and chemical damages to DNA or RNA form a large set of diseases, including many cancers, neurofibromatosis, muscular dystrophies, Parkinson disease, etc. 1 There is therefore a great interest in manipulating with precision nucleic acids (i.e. replacing, correcting, inserting, deleting). A number of nucleic acids have emerged as potential drugs such as pDNA for "Gene Therapy", 1 siRNA for "Silencing Therapy", 2 mRNA, up to the now-famous gene editing tools that include CRISPR-Cas9. However, nucleic acids cannot enter cells without help, which makes this delivery issue a central challenge in the field of gene therapies. Therefore, the big question regarding the translation of these nucleic acids into real drugs is: how to effectively and safely deliver nucleic acids to fix genetic malfunctions?

At present, viruses are most often used as vectors because they still are most effective. However, they also pose serious safety concerns due to their immunogenicity/genotoxicity, 3 not to mention their high cost of production and their narrow range of application with respect to the type of nucleic acid they can carry and the cell types onto which delivery is effective. Artificial alternatives are therefore much desired but despite decades of work they still remain elusive as an universal, safe and cheap solution to this big challenge. Generally, non-viral vectors are synthetic materials, based on amphiphilic cationic lipids, 4 cationic polymers, [START_REF] Pack | [END_REF][6][7][8] and their subsequent assemblies into micelles, liposomes etc, that can electrostatically recognize nucleic acids and transport them into cellular environment. 6,7,9,10 For instance, Lipofectamine ® is a commercially-available cationic lipid that is nowadays commonly used in laboratories around the world, yet, like many other applications in the clinic is still hampered by its toxicity. 11 Meanwhile, cationic polymers came out thanks to their ease of preparation, accessible structural diversity and good ability to condense nucleic acids. [START_REF] Laemmli | Proc. Natl. Acad. Sci[END_REF] Rapid progresses in cationic polymers provided safer systems for effective nucleic acid transfection, although still failing for translation to the clinic because of toxicity issues. 11,[START_REF] Laemmli | Proc. Natl. Acad. Sci[END_REF] Dynamic Combinatorial Chemistry (DCC) [START_REF] Corbett | [END_REF][START_REF] Reek | Dynamic Combinatorial Chemistry[END_REF][START_REF] Miller | Dynamic Combinatorial Chemistry In Drug Discovery[END_REF][16][17][18][19] enables access to large chemical libraries starting from molecular building blocks that reversibly interact with each other through reversible covalent reactions. 20,21,[START_REF] Lehn | Proc. Natl. Acad. Sci[END_REF] This means that dynamic covalent systems, formed through multiple reversible covalent bonds, can adapt their structure in response to physico-chemical stimuli. [START_REF] Lehn | [END_REF][24][25][26][27] Over the last decade, DCC has led to the development of Dynamic Covalent Polymers (DCP) which have attracted a great interest in the material chemistry community for their self-healing and adaptive properties. [START_REF] Mukherjee | Dynamic Covalent Chemistry: Principles, Reactions, and Applications[END_REF][START_REF] Imato | Dynamic Covalent Chemistry: Principles, Reactions, and Applications[END_REF][START_REF] Garcia | [END_REF][31][32][33][34][35] Currently, there is also a greater interest in the potential of such dynamic materials for nucleic acid delivery, motivated by the alluring possibilities to identify more effective vectors through dynamic combinatorial screening where building blocks compete and only the best fitted DCP binder is expressed when the target of choice is present. The ability to adaptively control the spatial distribution of interchangeable recognition groups on the dynamic surfaces of such reversible materials, may induce a high level of correlativity of synergetic interaction with the nucleic acid and the membrane barrier. In other words, this leaves the nucleic acid to template and to self-generate the fittest DCP material, for its own compaction. While this may represent a universal approach to identifying gene delivery vectors, it also bears the advantage of producing vectors self- degradable linkers and co-polymers that feature multiple groups involved in nucleic acid complexation, cell penetration, and endosomal escape (Fig. 2a). [START_REF] Geihe | Proc. Natl. Acad. Sci[END_REF][START_REF] Putnam | Proc. Natl. Acad. Sci[END_REF] However, since an ideal design is impossible to achieve a priori through a rational approach, robot-assisted combinatorial screening with automated polyplex preparation can be implemented in multiple-well plate formats in order to decipher structural aspects and find better and safer polymer vectors for gene transfection (Fig. 2b). [START_REF] Rinkenauer | [END_REF]70 Although costly, this approach enables a more rapid screening of different types of polymers on different cell types based on a range of criteria such as complex size determination, DNA binding affinity, polyplex stability, cytotoxicity, and transfection efficiency using optical assays. As an alternative exploiting reversible covalent chemistry, DCC offers the advantage that polymers do not need to be pre-assembled but it is precisely the binding to nucleic acids and/or the transport of the polyplex inside cells that amplify the formation of the best suited polymer vector from a pool of competing monomers. While such an approach has already been successful in medicinal chemistry programs, 71 it is true that its implementation in delivery applications remains more challenging. However, the recent demonstration by Alfonso et al. of DCC carried out on live cells represents a milestone achievement in this direction. 72 In a DCC approach, the dynamic platforms result by (molecular/supramolecular) self-assembly of specific core-connectors with the constitutive segments or the functional bio-interacting sites. The reversibility of the interactions between the components may Please do not adjust margins Please do not adjust margins finely and adaptively mutate the positioning of the components in the chemical space, resulting in the selfformation of the most adapted platform in the presence of DNA target and bilayer cell membrane environment. 73,74 Intricate dynamic configuration can provide much more active molecular, supramolecular or polymeric assemblies, making it possible to adapt an optimal structure for gene delivery at different dimensional levels. This emerging approach has hitherto been explored on three different systems: i) dynamic amphiphilic transfectors (Fig. 2c), 75 dynamic covalent polymers (Fig. 2d), 76 and dynamic constitutional frameworks (Fig. 2e) 77 which will be discussed in more details in this review.

Design criteria for positively-charged molecular building blocks

Cationic binding groups promote nucleic acid complexation via electrostatic interaction with the phosphodiester groups, which results in charge compensation and thus triggers a subsequent condensation into a nanoparticle 78 protected from enzymatic degradation. 36 However, cytotoxicity and rapid hemolysis is also related to cationic density such that great care must be taken when selecting the nature and number of This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins positively-charged compounds commonly inserted in cationic polymers for gene delivery cationic groups to be inserted within the polymer vector. 79,80 Common cationic compounds, including polyethyleneimine, 81,82 guanidinium 83 and amino acids 84,85 have been used for the construction of cationic polymers (Table 1).

As a typical cationic polymer, branched polyethyleneimine (PEI), having a series of primary, secondary and tertiary amines groups, displays favourable electrostatic adsorption to DNA, and shows buffer ability that promote endosomal escape. 81,86,87 Their divergent synthesis enables tuning the number and density of branching points, and varying the ratio between the three different types of amine. 81,88 A wide range of molecular weights, highly positive charges, topological structures and hydrophilic ability of PEIs, are critical facets that influence the transfection efficiency as well as the blood circulation time. 89,90 Different studies have proved that higher molecule weight PEI (≥25 kDa) are effective gene delivery vectors but could lead to undesirable cytotoxic effects caused by aggregation behaviours, while smaller molecular weight PEI (≤2000 Da) could exhibit limited transfection efficiency. 91,92 Guanidinium cations are very strong binders of negativelycharged oxoanions due to their high pKa (≈13) which make them permanently protonated in biological media. The introduction of cationic guanidinium groups can be achieved by guanidinylation 93,94 or by using off-the-shelf arginine as a guanidinium-containing amino-acids. 67,95 Arginine-containing compounds interact very well, via salt-bridge interactions, with the phosphodiesters of nucleic acids -an effect which has been coined "arginine magic". 96,97 The length, number and density of positive charges are key factors when using oligoarginine conjugates in gene delivery. 98 Some artificial derivatives of guanidiniums such as the guanidiniocarbonyl pyrrole motif show improved oxoanion binding and thus bears a strong potential for nucleic acid delivery applications. [99][100][101] Histidine also can be grafted to cationic polymers. Thanks to the low pKa of its imidazole ring(≈ 6), it often partake in an acid-dependent fusion and leakage of liposomes. 102 The high loading of histidine into cationic polymers effectively promote efficient endosomal escape but can also sometimes be deleterious to the nucleic acid complexation -the benefit to the overall transfection efficiency being affected by the casedependent relative importance of those two steps. [103][104][105] In contrast, although lysine-containing cationic polymers are better at nucleic acid complexation and cell penetration, 106,107 they do not display effective endosomolytic activity, thereby causing low transfection efficiency. 104,108

Reversible reactions suitable in gene delivery applications

Dynamic constitutional chemistry rests on reversible covalent reactions.

Various reversible reactions, such as amino/carbonyl-imines, sulphides/disulphides, Diels-Alder reaction, alkene and alkyne metathesis, have been applied to design dynamic covalent polymers as adaptive functional materials. [START_REF] Corbett | [END_REF]61,[109][110][111][112][113][114][115] The reversibility of dynamic linkages endow polymers with attractive functionality, for example selfhealing, stimulus-responsiveness, degradability, recyclability and replasticity. Despite such prominent developments of DCPs in the field of material science, the application in gene delivery is a much more recent adventure which bears distinct challenges. For instance, considering the unique conditions that exist in biological environments, adjustment and rearrangement of reversible linkages must be compatible with an aqueous medium and must operate at room temperature with appropriate thermodynamic and kinetic features at the typically low concentrations used. Ideally, the chemoselective reversible reaction should be biorthogonal, but the groups of Matile and Adibekian have interestingly shown that a transient cross-reaction with biomolecular constituents of the cell membrane can assist cell penetration (vide infra). 116 To date, carbonyl condensation reactions, disulphide bond, boronate ester remain the most popular choice (Fig. 3). Please do not adjust margins Please do not adjust margins One of the most popular chemistries used for the construction of scaffolds for gene transfection is the iminetype condensation reaction between carbonyls (aldehyde or ketone) and primary amine groups. Playing with the α-effect give access to different types of conjugates, such as imines, oxime, hydrazones and acyl-hydrazones depending on the selected nucleophile, which have different thermodynamic stabilities in aqueous media and kinetics of formation. 117,118 Imines are widely used because of straightforward synthesis, but they usually revert back to carbonyl compound and primary amine too quickly in aqueous media. 119 Research on their hydrolysis have shown that oxime linkages presents better performance on hydrolytic stability compared to imines. 117,120 Sitting somewhere in between, acyl-hydrazones are of interest since they show sufficient stability while remaining dynamic on a reasonable time-scale (typically hours to days depending on the conditions). Placing assisting groups next to the reactive moieties has been shown to modulate the stability and kinetics of formation and exchange reactions, 121- 124 allowing fine tuning those conditions by molecular engineering. For example, an ortho boronic acid substituent for aryl ketones can significantly stabilize the corresponding imines at neutral pH (Fig. 4), while its conjugation with phenylhydrazine takes place with rate constants as high as 10 3 M -1 s -1 . 122 Alternatively, nucleophilic catalysts can also be used. [125][126][127][128][129][130] Importantly for nucleic acid delivery where endosome escape is a common issue that can be addressed using pH-responsive systems, acyl-hydrazones are acidsensitive and are typically hydrolysed at acidic pH (pH ≈ 4-6). Finally, all those C=N groups may feature double dynamic processes, configurational and constitutional dynamics, 131,132 which in principle can be used to provide an handle over their biological activity. 133 Disulphide bond is triggered by thiol oxidation and this redoxcontrolled reversible process plays a great role in stabilizing the 3D-folded of proteins. Disulphide exchange require thiol deprotonation and the formation of an active thiolate anion making this chemistry adaptive at basic pH. 134,135 Disulphidebased dynamic carriers have been exploited in the design of nucleic acid delivery carriers, 136,137 where glutathionemediated biodegradation can be applied to facilitate gene release from polyplexes cargo. This method provides a safe and efficient gene delivery in the intracellular compartment where the concentration of glutathione reaches the mM range. 138 As further discussed below, disulphide exchange reactions have also been shown to take place in situ with constituents of cell membranes.

Boronate esters can be formed though reversible condensation reactions between boronic acids (i.e. boric or phenylboronic acid) and vicinal diols, especially cis-1, 2-and 1, 3-diols. At present, and despite their popularity as reversible covalent bond, boronate esters have received little interest for making dynamic covalent polymers for gene delivery, most likely due to their limited association constants in aqueous media. Nevertheless, boronic esters can be interestingly used to break down polyplexes -though oxidization triggered by reactive oxygen species in physiological condition -and then release nucleic acid. 139,140 A tailored dual-responsive gene delivery vehicle was formulated via boronic acid chemistry, displaying appreciable endosome escape and rapid gene liberation. 140 Please do not adjust margins Please do not adjust margins One of the early contributions to gene delivery using dynamic covalent chemistry was the development of dynamic cationic amphiphiles (Fig. 5a) that are self-assembled by DCC through the direct in situ conjugation between cationic heads and lipophilic tails. Then, capitalizing on the multivalent binding often expressed by cationic polymers, different architectures of cationic dynamic covalent polymers -from linear to 2D and 3D -have been explored for gene delivery: i) linear dynamic polymers (Fig. 5b), ii) side-chain dynamic polymers (Fig. 5c), and iii) dynamic constitutional frameworks (Fig. 5d).

Architectures of cationic dynamic covalent polymers

Dynamic cationic amphiphiles

Amphiphilic cations have been designed to complex anionic nucleic acids and the cellular uptake capacity of the counteranion-activated DNA have been demonstrated. 67,83,141 For example, calixarene macrocycles have been used to combine multiple guanidinium cations to form counteranion activators as cation transporters. 83 The conical architecture of calixarenes provided a wide space to spatially fix multiple cationic substituents around the rim of the unique macrocyclic structure. Tetra-or hexa-guanidinium cations with diverse amphiphilicity were used to complex DNA. Beyond this, dynamic amphiphiles complexes which are constructed by combining charged heads with hydrophobic tails through reversible linkages (Fig 5a), 75 are capable of mediating nucleic acids as well as of penetrating through lipid bilayer membranes, representing a privileged choice for achieving cellular uptake and gene transfection. 96,142,143 Moreover, the appropriate selection of the reversible covalent reaction can impart redox or pH sensitivity to the amphiphilic self-assembly and, thereby, a control over the delivery of nucleic acids.

Lehn and co-workers reported early on an example of dynamic amphiphile where the cationic head and the lipophilic tail is connected through a single pH-sensitive acyl-hydrazone bond. 144,145 Matile and coworkers reported dynamic octopus amphiphiles, (Fig. 6) composed of guanidinium cation "head" and hydrophobic "tails". 146,147 In these systems, tetrahydrazides as hydrophilic counterions provided fourtailed octopus amphiphiles to multivalently bind the nucleic acid. Hydrazone, oxime and disulphide linkages were used to expand their library by means of various dynamic cationic amphiphiles with double charged heads and four hydrophobic tails. 75 These multi-tails counterion transporters were synthesized for the activation of DNA and a comprehensive screening of these dynamic polyion-counterion complexes showed potential application toward sensing 148 and cellular uptake. 142,149 Dynamic cationic amphiphiles are attractive multicomponent delivery platforms that combine the advantages of i) cationic hydrophilic head groups for condensation of genes, ii) membrane compatibility of hydrophobic tails, and iii) reversible covalent bonds that endow responsiveness. Structure-activity relationships can be elucidated through fragments screening and in situ self-assembly of structurallydiverse components constituting the cationic amphiphiles. By means of chemoselective reversible covalent bonds that can be formed in situ, dynamic cationic amphiphiles can be constructed to quite large and complex libraries with hundreds of combinations. A large library with 900 dynamic cationic amphiphiles was generated using 18 heads and 50 tails, leading to the identification of 160 candidates out of the 900 possibilities which were taken further for cellular uptake as "artificial noses" for siRNA delivery. 150 These dynamic cationic amphiphiles present great abilities to complex nucleic acids and transfect them through cell membranes with high reproducibility and low toxicity. A moderate number of dynamic cationic amphiphiles were generated for rapid identification and screening, thereby this methodology based on dynamic cationic amphiphiles presents a considerable potential for gene delivery.

Linear dynamic polymers-dynamers

Linear dynamic polymers are formed by the reversible association of ditopic monomers, and thus contain the reversible linkage in their main chain. This feature makes them dynamic not only in terms of incorporation/decorporation of monomers but also in terms of chain extension and contraction.

The group of Seymour reported early on an elegant strategy using disulphide formation as reversible covalent reaction on cationic peptides end-terminated with cysteines at both N and C termini. Oxidative polymerization lead to cationic polymers able to complex DNA and to undergo intracellular reductive depolymerization. 152 The delivery and controlled release intracellularly of gene in live cells has been carried out by using cell-penetrating poly(disulphides) (CPDs), this can provide an efficient solution to minimize cytotoxicity in the field of gene transfection. 153 Matile's group have exploited dynamic cationic linear CPDs synthesised through ring-opening disulphide exchange. 154,155 In their design, guanidinium cations were first covalently connected to a strained disulphide, linear carriers were grown by substrates-mediated polymerization subsequently (Fig. 7). 151 The implantation of disulphide ring may facilitate thiol-mediated cellular uptake and achieve controlled release of targets by reductive depolymerization. 116,156,157 Indeed, those CPDs rapidly depolymerize by monomer removal through self-cyclization within minutes after intracellular reduction, and their variable length has been shown to determine their cellular localization. 155,158 Glycosylation with -D-glucose, -Dgalactose, D-trehalose, and triethyleneglycol has been later used to increase the solubility of classical CPDs and to achieve multifunctional cellular uptake. 159 The results indeed indicate better cell uptake in the cytosol and nucleus together with a reduced cytotoxicity, the CPD bearing -D-glucose being the most active. Also, a role for the sugar-receptor interaction at the cell surface in the cell penetration process has been evidenced by competition experiments, cell penetration being hampered by large excess of D-glucose but not with L-glucose.

Ulrich and co-workers have synthesised linear dynamic covalent polymers though a polycondensation process exploiting acyl-hydrazone linkages (Fig. 8a). 76 The first generation of these linear hybrid dynamers was designed to combine guanidinium-based or ethyleneimine cationic groups with short ethylene-glycol chains. The results show that oligomers do indeed self-assemble when the polycondensation reaction is carried out at high concentration (typically > 50 mM), and that the resulting dynamers display multivalent binding to plasmid DNA in aqueous buffer as well as in biological serum. 160 Moreover, the acyl-hydrazone-based dynamers have been shown to be pH-sensitive and to undergo degradation at pH ≈ 5 corresponding to the pH of late endosomes, while remaining stable over much longer period of time at physiological pH. The second generation of linear dynamers includes modified amino acids and involve acyl-hydrazone and oxime condensation reactions for polymer formation (Fig. 8b). 161 This more versatile synthetic approach enabled testing different modified amino acids (Arg, Hys, Lys) and arginine stood out for imparting better binding to DNA. Combination of the pHsensitive acyl-hydrazone reversible covalent bond with the redox-sensitive disulphide bond could also be achieved for making dual-responsive dynamers. The results obtained through fluorescence displacement assays and gel electrophoresis demonstrate good complexation not only to long dsDNA but also to short siRNA. Biological experiments on live cells reported an effective transfection of siRNA -even surpassing lipofectamine -with limited cytotoxicity. This journal is © The Royal Society of Chemistry 20xx

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Side-chain dynamic polymers

Side-chain dynamic polymers are made of non-dynamic polymers connected to multiple functional side-chains through reversible reactions such as acyl-hydrazone 162 and disulphide. 152 The length of side-chain dynamic polymers is therefore imposed by the structure of the polymer scaffold and it is their decoration which is susceptible to adaptation by constitutional exchange processes -the number and positions of those side-groups being imposed by the number and position of the reactional handles introduced in the polymer scaffold. [164][165][166] Fernandez-Trillo, Montenegro and co-workers used poly-acryloyl polymer scaffolds bearing multiple hydrazide groups to prepare amphiphilic vectors through a covalent self-assembly approach (Fig. 9). 162,163,[167][168][169] Polymers of different lengths were functionalized, via acyl-hydrazone chemistry, with varying ratio of cationic and lipophilic aldehydes. Subsequently, a rapid and effective in cellulo screening enabled the identification of the best nucleic acid vectors.

Montenegro's group then exploited a helical linear peptide as pre-organized scaffold presenting lipophilic and cationic hydrophilic faces (Fig. 10). 163 Two reactive hydrazide connectors were placed on this main chain and the same dynamic post-functionalization approach was implemented using a library of hydrophobic aldehydes. It was found that the lipophilic side-chain affected the secondary folding of the peptide scaffold and its overall amphiphilicity, thereby strongly affecting subsequent interactions with nucleic acids and cell membranes. In the end, the screening was successful and led to the identification of an effective vector of plasmid DNA. More recently, the Montenegro group reported the design, synthesis, and biological evaluation of glycosylated cellpenetrating peptides based on their original design of sidechain dynamic polymers. 170 Two identical glycans such as -Dmannose, -D-glucose, N-acetyl--D-glucosamine, -Dgalactose, and a branched trisaccharide of -D-mannose were anchored onto the peptide through oxime ligation. Excellent cell penetrating activities have been observed with improved uptake efficiency/cytotoxicity balance. Unfortunately, the screening of different glycans indicate that cell penetration is mainly imparted by the peptide and not by the glycan residue, thereby limiting for the moment the potential of this approach for achieving cell-selective delivery.

Dynamic constitutional Frameworks -DCFs

Examples of reversible cross-linking of cationic peptides have been reported early on using disulphide chemistry. [171][172][173] More recently, imine-based Dynamic constitutional frameworks -DCFs have been proposed by Barboiu and co-workers for adaptive gene recognition and cell-transfection. 174,175,176 Poly(ethylene glycol)-bis(3-aminopropyl) terminated, spermine and Jeffamine were used as connecting building blocks and connector centres, linked together by labile, reversible Please do not adjust margins Please do not adjust margins covalent bonds to form cross-linked 3D scaffolding platforms. Cationic blocks have been attached in the head of scaffolds to form hydrophilic corona, giving them the ability to electrostatically bind nucleic acids. Importantly, they undergo exchange, incorporation/decorporation of their subunits, synergistically interacting and adapting the overall 3D nanostructure in the presence of DNA and bilayer membrane environment. The core scaffolds used for the formation of the DCF are multivalent platforms with multivalent binding affinity towards nucleic acids. This might play an important role in the ability to finely mutate and adaptively implement reversible rearrangements of the components, toward a high level of correlativity of their hypersurfaces in interaction with the DNA and the cell membrane barrier. This means that gene biotargets can self-select their optimal and functional DyNAvector from a mixture of reversible 3D DCFs. The DCF strategy showed an easy and efficient identification and rapid screening for DNA recognition and transfection. The DCFs are highly soluble in water and mainly positive charged when in physiological conditions, to increase the possible interaction with the negatively charged phosphates groups on the DNA. The incipient development of DCFs have been described by Barboiu and co-workers in 2015 showing that PEG macromonomers, trialdehyde core connectors and positively charged heads can be used to generate DCFs for DNA recognition (Fig. 11). The ability of DCF1-5 to bind negative dsDNA was evaluated using the agarose gel retardation assays and DNA-binding affinity is related to the greater capacity of the guanidinium vs. ammonium group to interact with phosphodiesters of DNA. [START_REF] Corbett | [END_REF] The simplicity and robustness of the synthetic strategy allow rapid screening of conditions for generating systems with optimal synergistic DNA binding affinities.

The next generations of DCFs was designed in order to obtain good transfection efficiency and a low cytotoxicity. 176,177 Barboiu et al. described the synthesis and characterization of a class of PEI based DCFs, co-self-assembled with PEG components and leading to adaptive spatial distributions in the presence of interacting DNA biotargets (Fig. 12). 177 PEI is one of the most studied synthetic cationic DNA vectors and branched PEI of high molecular weight is considered to be one of the most efficient gene carrier for plasmid DNA, oligonucleotides and small interfering RNAs (siRNA)s. The high transfection efficiency for PEI/DNA polyplexes is attributed to the unique capacity of PEI to buffer endosomes. However, the use of PEI in vivo and in vitro gene delivery is limited because of its low colloidal stability and its considerable cytotoxicity. To enhance stability and biocompatibility of PEI polyplexes, they can be combined with PEG; however, PEGs shield the positive charges of PEI, which often has the undesired effect of decreasing transfection efficiency. The PEI-based DCFs are able to act as vectors, by forming stable polyplexes with dsDNA ranging between 40 and 125 nm. All tested vectors were capable of transfecting DNA into HeLa cells and demonstrated low cytotoxic levels; even at an N/P=200 cell viability is over 90%. We can conclude that the presence of the PEG component and a moderate amount of b-PEI in DCFs are both important in the construction of highly transfecting and cytofriendly polyplexes, well tolerated by cells. 177 Reversible recombination of cationic heads with hydrophilic and hydrophobic DCF-constitutive building blocks result in the formation the multivalent core-shell DCF architectures with an optimal diameter of 100 nm that may be adaptively generated in the presence of DNA targets (Fig. 13). 178 The fittest DCFs simultaneously exhibit optimal DNA binding, superior transfection yield to standard transfection SuperFect agent and preserve high HEK 293T cell viability. 176 Similarly a multivalent polycationic structure with aromatic-guanidinium heads may be generated through imine bridges between 1,3,5benzenetrialdehyde and tri(2-aminoethyl)amine. This provides an alternative method to design polymeric polyplexes for gene delivery. 77 Adaptive DCF vectors can be synthesized via constitutional self-assembly of PEG and squalene components with bPEI cationic binding groups with the core centers around the DNA biotargets (Fig. 14). They adaptively generate micellar polyplexes with variable sizes that transfect HeLa cells and proved low cytotoxic levels. Using different molar and N/P ratios among components or in combination with DNA, Please do not adjust margins Please do not adjust margins spherical and tube-liked core-shell particles can be synergistically obtained as showed by transmission electron microscopy. 179 The polyplexes size and polydispersity are close related to transfection efficiency of 12% at N/P=50 that can be obtained with particles < 200 nm diameter and a narrow polydispersity, while presenting a minimal toxicity. 179

Toward universal gene delivery through adaptive self-assembly

The unique feature of dynamic covalent polymers, compared to traditional and degradable polymers, is their capacity to adapt throughout the multi-step gene delivery process.

Adaptation to nucleic acid binding

Adaptation of dynamic covalent polymers to nucleic acid is an interesting approach toward identifying vectors that are selffitted for one particular application (e.g. pDNA, siRNA, mRNA delivery). Interestingly this specificity does not limit the versatility of the approach since the adaptive behaviour enables in situ reconfiguration of the dynamic polymers to a different templating nucleic acid. Early examples provided proof-of-concepts for the use of dynamic combinatorial chemistry on nucleic acid targets 180 but few of them addressed the application of gene delivery. In this line, we have reported constitutional adaptation of low molecular weight vectors to nucleic acids. 84 Using dynamic covalent polymers, the group of Aida reported a major discovery in the field in 2015. 181 They studied water-soluble telechelic dithiol monomers bearing multiple (2-4) guanidinium groups that are separated by a 7.4 Å ether spacer, fitting the regular separation of 6-7 Å between neighbouring phosphodiesters in nucleic acids (Fig. 15). Oxidative polymerization of TEG Gu4 was carried out in the presence of siRNA using KI/I2 as oxidant, and gel electrophoresis evidence siRNA complexation at N/P>20 only in those oxidative conditions, while the subsequence reduction using GSH triggered siRNA release after 1 min incubation. TEG Gu3 showed a similar behaviour, whereas the others led to the formation of a precipitate when mixed with siRNA. Physico-chemical studies (TEM, DLS) reveal the formation of "nanocaplets" with diameters of < 10 nm. Dialysis was used to remove the excess of unbound cationic monomers/polymers, and the numbers of bound TEG Gu4 was determined by an Ellman's assay, which confirmed a 1:1 guanidinium:phosphodiester binding stoichiometry, thereby supporting the model of a siRNA-templated polymerization (Fig. 16). Alternatively, the polyplex was incubated with ZnCl2 for selective hydrolysis of siRNA and the unbound polymers were subsequently analysed by LC-MS and revealed the formation of the hexamer of TEG Gu4, understandable in terms of charge compensation (24 positive charges in the hexamer vs. 21 negative charges per siRNA strand). All those results firmly demonstrate that oxidative polymerization takes place, and that siRNA templates the formation of the resulting polymers. Satisfyingly, this approach was successfully implemented for gene silencing using a luciferase assay on human hepatocellular carcinoma Hep3B cells. While the results reported are very encouraging, rivalling the activity of the commercial transfection reagent DharmaFECT although the amount of uptaken siRNA was five-fold smaller, the authors noted that the "rather poor cellular uptake activity can be improved when the nanocaplet is connected to arginine peptide-Arg6", which could pinpoint to a limitation of such electrostatically-driven siRNA-templated approach leading by charge compensation to rather neutral polyplexes whereas a net positive charge is often desired for promoting cell penetration. A solution to this issue may to be insert ligands that promote cell uptake and this has recently been realized by the same group using transferrin which was non-covalently attached to the surface of the nanocaplet. 182 Finally, while nucleic-acid templated polymerization can also be applied to classical non-dynamic polymers, 183 it is precisely the reversible character of DCPs that will subsequently enable them to adapt their constitution throughout the delivery process as further discussed hereafter.

Adaptation to membrane binding

The cell membrane represents a physical barrier, made of chemical constituents that dynamically rearrange depending on the conditions, 184 that polyplexes have to cross in order to reach the cytosol. The so-called "tryptophan power", 185 which was found when Trp amino acids are inserted within cellpenetrating peptides, illustrates a simple way, among other lipid-based delivery approaches, 37 to improve the interaction with cell membranes in order to favour translocation. A key point in designing amphiphilic systems is the hydrophobic/hydrophilic balance and their three-dimensional presentation. Dynamic combinatorial chemistry has already been implemented on simple models to identify supramolecular transporters of ions, 186,187 amino acids, 188 toward adaptive separation membranes. 20,35,189,190 Apart from the latter examples, works studying dynamic covalent polymers at membrane interface remain rare. Here the situation is much more complex to investigate due to multiple dynamics possibly taking place. Indeed, in addition to the constitutional dynamics described above that operate through incorporation/decorporation of monomers, dynamic covalent polymers are also involved in ring-chain equilibria. All these factors may strongly affect the translocation efficacy since, for instance, it is now well established that cyclic TAT cell penetrating peptides and cyclic PEI enter cells more effectively than their linear counterparts. 191,192 Interestingly, the group of Sagan demonstrated that thioldisulphide dynamic covalent exchange reactions with cellsurface thiols can take place at the cell membrane, which was considered as a new route toward cell entry by cellpenetrating peptides exploiting a transient covalent bond. 193 Using peptides appended with a single asparagusic acid residue, the groups of Matile and Adibekian have interestingly shown that such a transient cross-reaction with biomolecular constituents of the cell membrane can indeed assist cell penetration (Fig. 16). 116 Proteomics experiments revealed that the transferrin receptor is the biomolecular target onto which disulphide bonds establish with two cysteines following an exchange reaction. Cellular uptake was later found to be even more active with 1,2-diselenolanes compared to 1,2dithiolanes, 194 with recent improvements using benzopolysulfane networks. 195 polymersomes). 196,197 More recently, the Matile group disclosed the improved cell-penetrating capacities of benzopolysulfanes which form a complex dynamic-covalent network of extreme sulfur species, including cyclic oligomers, from dimers to heptamers. 198 Biscysteine-bearing peptides were also found to be excellent at cell penetration, rivalling with the "Arginine Magic", thanks to their ability to establish multiple disulphide bonds with the constituents of the cell membrane. 199,200 Overall, the results underscore the importance of studying dynamic covalent chemistries at the cell membrane interface in order to provide new ways to effectively enter cells. 39 Undoubtedly, such new knowledge will strongly benefit the development of dynamic covalent polymers for gene delivery.

Summary and outlook

The search for synthetic polymer gene delivery vectors has been underway for a couple of decades now. The variability of nucleic acids of pharmaceutical interest has grown, along with the large number of cells to be transfected, which reinforce the interest in versatile, rapid and cheap discovery methodologies. While the rational design approach has been instrumental it does not fulfil those criteria and combinatorial screening represents a useful tool to streamline this process. Going one step further, constitutional dynamic approaches enable adaptive self-assembly to take place, leading potentially to the self-selection of the best fitted vector. Dynamic combinatorial screening is thus a simplified method Please do not adjust margins Please do not adjust margins to generate the most effective vector from complex dynamic libraries of interconverting species such as dynamic covalent polymers. Herein, we reviewed the progresses made in that direction and highlighted recent results obtained using dynamic covalent polymers for gene delivery. We summarized the current challenges that have to be addressed in gene delivery, described the structural and functional features that should be considered when designing dynamic covalent polymers, presented the reversible covalent reactions suitable for this application, and discussed in details the recent results that have been achieved using different architectures of dynamic covalent polymers: i) dynamic amphiphiles, ii) linear dynamic covalent polymers, iii) side-chain dynamic polymers, and iv) dynamic constitutional frameworks. Dynamic amphiphiles complexes are composed of charged hydrophilic heads and hydrophobic tails through reversible linkages. Linear dynamic polymers show multivalent nucleic acids complexation correlated to the dynamic self-assembly of their main-chain backbone. Side-chain dynamic polymers can hook functional molecules and achieve responsive release and endosomal escape in live cells. Dynamic constitutional frameworks DCFs allow to self-adapt and self-generate the optimal 3D architectures for gene therapy. This dynamic covalent chemistry-based strategy avoids tedious multi-step synthesis of complex compounds, instead it exploits a covalent self-assembly process to easily generate numerous constituents that can be screened and lead to the identification of effective nucleic acid vectors having high complexation ability, good transfection efficiency, and limited toxicity.

Finally, we underscored the unique feature of such a dynamic constitutional that offer great perspectives toward universal gene delivery through adaptive self-assembly using selected examples that demonstrate in situ dynamic covalent polymer formation templated by nucleic acids and their dynamic reorganization when interacting with cell membranes. The examples presented here forming the basis of this area arise mainly from parallel exploratory works studying different type of systems (different architectures, different types of positive head groups and reversible covalent linkages) and it will be important in the future to establish structure-reversibilityactivity relationships for gaining a better understanding that will help shape the future of the field. This is just the beginning of the story and much remains to be investigated to turn it into successful applications. For instance, in situ self-assembly and adaptation has been characterized in just a few systems to date, and constitutional adaptation to different types of nucleic acids and cells has to be demonstrated to validate the versatility of this approach. There, constitutional adaptation through, for instance, clustering can also be harnessed for an adaptive multivalent binding 48,[201][202][203] to cell membrane receptors that, in turn, should promote (selective) cell uptake. A greater control and/or fine tune of the dynamic response should also be sought.

Translating those interesting proof-of-concepts into real therapeutic applications will also require testing in vivo (e.g. zebrafish model, or mice) gene delivery using dynamic covalent polymers. Last but not least, while dynamic and degradable systems are theoretically attracting and potentially less toxic, managing the safety aspect of such dynamic systems, where interconversion between multiple species is possible, will be tricky part. But the perspectives of finding finally universal yet self-fitted synthetic gene delivery vectors through such cheap and versatile process are worthwhile the effort. This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins This journal is © The Royal Society of Chemistry 20xx

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Fig. 1

 1 Fig. 1 Barriers in the nucleic acid delivery pathway of polyplexes. 36 a) nucleic acids complexation, b) systemic circulation of polyplexes in blood, and c) cellular barriers encountered by polyplexes during cell internalisation. Reproduced with permission from ref. 36. Copyright 2015 American Chemical Society.

Fig. 2

 2 Fig. 2 Strategies for identifying polymer vectors for gene delivery: a) rational design of degradable and multifunctional block copolymers, b) high-throughput combinatorial screening, c) dynamic combinatorial transfectors based on cationic amphiphiles, d) dynamic covalent polymers, and e) 3D dynamic constitutional frameworks.

Fig. 3

 3 Fig. 3 Reversible covalent reactions: (a) amino/carbonyl condensation, (b) sulphide/disulphide and (c) boronate ester reactions.

Fig. 4

 4 Fig. 4 (a) Effect of ortho boronic acid substituent on imine formation at neutral pH; and (b) mechanism proposed for explaining the rapid formation of iminoboronates derived from α-nucleophiles.

Fig. 5

 5 Fig. 5 Architectures cationic dynamic covalent vectors used in gene delivery: (a) dynamic amphiphiles, (b) linear dynamic polymers, (c) side-chain dynamic polymers, and (d) dynamic constitutional frameworks.

Fig. 6

 6 Fig. 6 Covalent self-assembly of octopus amphiphiles for polyion transport. 147 Reproduced with permission from ref. 147. Copyright 2010 WILEY-VCH.

Fig. 7

 7 Fig. 7 Formation of cationic linear cell-penetrating poly(disulphide)s vectors by ringopening polymerization. 151 Reproduced with permission from ref. 151. Copyright 2013 American Chemical Society.

Fig. 8

 8 Fig. 8 Dynamic covalent assembly of linear dynamic covalent polymers for DNA complexation and siRNA delivery. 21 Reproduced with permission from ref. 21. Copyright 2019 American Chemical Society.

Fig. 9

 9 Fig. 9 Dynamic functionalization of polyhydrazide polymers by cationic and lipophilic aldehydes for identifying amphiphilic vectors of nucleic acids. 162 Reproduced with permission from ref. 162. Copyright 2016 WILEY-VCH.

Fig. 10

 10 Fig. 10 Peptide-scaffolded side-chain dynamic covalent polymers for the intracellular delivery of plasmid DNA, with wheel diagram representing the helical conformation of facial amphiphilic peptide scaffold P1. 163 Reproduced with permission from ref. 163. Copyright 2017 Royal Society of Chemistry.

Fig. 11

 11 Fig. 11 Dynamic constitutional frameworks for DNA recognition; DCF synthesis combining PEG, 2 and 1,3,5 benzene-trialdehyde cores 1, resulting in the formation of neutral DCFs 1 and2, followed by treatment with positively charged heads to led to the final cationic DCFs 3-5. 174 Reproduced with permission from ref. 174. Copyright 2015 Royal Society of Chemistry.

Fig. 12

 12 Fig. 12 Dynamic constitutional frameworks containing PEI/PEG components for DNA recognition and transfection. 177 Reproduced with permission from ref. 177. Copyright 2015 Royal Society of Chemistry.

Fig. 13

 13 Fig. 13 Dynamic constitutional frameworks containing hydrophobic/hydrophilic components for DNA recognition and transfection: a) The proposed self-aggregation mechanism in the formation of DCF/DNA polyplexes; b) TEM image for corona DCF particles; c) AFM image and particle size distribution for DCF/plasmid pEYFP at N/P=100. 178 Reproduced with permission from ref. 178. Copyright 2015 American Chemical Society.

Fig. 14

 14 Fig. 14 Squalene-based self-assembly 9 results in the formation of DyNAvector nanoparticles binding DNA to form polyplex 10 used for gene delivery. 1,3,5-benzenetrialdehyde 1 core-connectors, Squalene-PEG 2 and PEG 3, segments and DNA binding sites bPEI 4. The intermediary units 5, 6 resulted after combination of components 1-3 have been combined with 4 to give multivalent charged unit, 7, 8. 179 Reproduced with permission from ref. 179 Copyright 2015 Royal Society of Chemistry.

Fig. 15

 15 Fig. 15 Chemical structures of dithiol monomers bearing multiple guanidinium groups (left), and principle of siRNA-templated polymerization.

Fig. 16

 16 Fig.16 Disulphide-based dynamic covalent chemistry taking place at the cell membrane and contributing to facilitating cell uptake of a drug through a transient covalent adduct with the transferrin receptor protein 1 TFRC.

Table

  Representative

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This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 9

Acknowledgements

DS appreciates receiving a scholarship from China Scholarship Council as support at University of Montpellier, France. SU thanks the ANR (ANR-17-CE07-0042-01) for funding.

Conflicts of interest

"There are no conflicts to declare".


Paula Ramos-Silva

Frédéric Marin
email: bfrederic.marin@u-bourgogne.fr

Proteins as functional units of biocalcification -an overview

Keywords: biomineralization, calcifying matrix, proteins, proteomics, sequences, functional domains

High-throughput approaches such as genomics, transcriptomics and proteomics have led to the discovery of a larger set of biomineralization genes than previously foreseen. These gene lists are often difficult to decode in light of the current models of calcification. Here we overview the proteins available in UniProt (Universal Protein Resource), that were identified directly in metazoan calcium carbonate mineralized structures or known to have direct key-functions in calcification processes. Functional annotation of the protein datasets using Gene Ontology reveals that functions like carbohydrate binding, structural and catalytic activities (e.g. hydrolase) are commonly represented across the organic matrices.

1.

Introduction

In the metazoan world, many organisms synthesize mineralized structures of calcium carbonate (CaCO 3 ) through a physiological process called biocalcification. A significant number of calcifying species lives in the aquatic environment and belong to distinct groups including sponges, corals, brachiopods, mollusks, crustaceans, echinoderms and chordates. A common feature among these groups of organisms is the production of CaCO 3 -mineralized structures by a biologically controlled mechanism. Indeed, this process is highly regulated at all stages of biomineral formation [START_REF] Lowenstam | On Biomineralization[END_REF]. In particular, it requires: [START_REF] Lowenstam | On Biomineralization[END_REF] the delineation of the mineralizing space by cell membranes or polymers;

(2) the formation of an array of macromolecules and its subsequent targeting to the site of mineralization; (3) the pumping of inorganic ionic precursors to set up a saturated environment; and finally (4), the control of certain proteins over crystal nucleation, growth and inhibition, providing a scaffold for mineral deposition. Although biologically controlled mineralization is present in microorganisms such as bacteria (magnetotactic [START_REF] Bazylinski | Controlled Biomineralization of Magnetite (Fe3O4) and Greigite (Fe3S4) in a Magnetotactic Bacterium[END_REF]) and unicellular algae (diatoms [START_REF] Kröger | Diatoms-from cell wall biogenesis to nanotechnology[END_REF], coccolithophores [START_REF] Marsh | Regulation of CaCO3 formation in coccolithophores[END_REF]) or protozoans (ciliates [START_REF] Lemloh | Genesis of amorphous calcium carbonate containing alveolar plates in the ciliate Coleps hirtus (Ciliophora, Prostomatea)[END_REF]), the process is undeniably more broadly represented in animals. Non-vertebrates in particular can produce a wide-range of external CaCO 3biominerals such as shells (in mollusks and in brachiopods), carapaces (in crustaceans), exoskeletons (in corals and sponges) and calcified tubes (in serpulid annelids), but also internal ones like spicules and spines (in sponges and echinoderms, respectively) and gastroliths (in crustaceans). In vertebrates, CaCO 3 -mineralized tissues are less common. The main examples refer to structures like fish otoliths [START_REF] Kang | Sparc protein is required for normal growth of zebrafish otoliths[END_REF] and otoconia [START_REF] Xu | Expression, functional, and structural analysis of proteins critical for otoconia development[END_REF], which primary function is to provide sense of gravity, balance, movement and direction, and eggshells that provide an outer hard protection to avian eggs [START_REF] Hincke | The eggshell: structure, composition and mineralization[END_REF]. It is common to describe biominerals as organo-mineral assemblages where the mineral, the major component, is embedded in a minor organic matrix. This latter is often a complex mixture of macromolecules -proteins, glycoproteins, polysaccharides and lipids -produced by the organisms and directly involved in the regulation of the mineral deposition. In most cases, the macromolecules are occluded within the CaCO 3 during its growth, constituting the "organic fraction" of the biomineral or the more generally called organic matrix (OM). Although there is still much to study about the exact mechanisms by which the components of the OM, in particular the proteins, control the formation of the calcified tissues, some key features of these proteins are recognized as being functionally important. The first common trait is the high content in aspartic acid and, in a lesser degree, glutamic acid. This feature is frequently observed in proteins of mollusk shells and coral exoskeletons [START_REF] Addadi | Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization[END_REF]. These proteins are recognized to have strong inhibitory function on the crystal growth and they are suspected to control the crystal morphology by selective adsorption on mineral surfaces [START_REF] Addadi | Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization[END_REF][START_REF] Gotliv | Mollusk shell acidic proteins: in search of individual functions[END_REF][START_REF] Gotliv | Asprich: A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina rigida[END_REF][START_REF] Mass | Cloning and Characterization of Four Novel Coral Acid-Rich Proteins that Precipitate Carbonates In Vitro[END_REF], due to their multiple negative charges. Another interesting feature of OM proteins are the post-translational modifications, in particular, glycosylations and phosphorylations, which can greatly contribute to the polyanionicity of such proteins [START_REF] Albeck | Polysaccharides of Intracrystalline Glycoproteins Modulate Calcite Crystal Growth In Vitro[END_REF][14][15][16]. Besides glycoproteins, the OM contains polysaccharides, which are important for structuring [17] the organic-mineral framework but also for interacting directly with the crystal surfaces [START_REF] Albeck | Polysaccharides of Intracrystalline Glycoproteins Modulate Calcite Crystal Growth In Vitro[END_REF]. Enzymes have also been identified in organic matrices, the best example being the carbonic anhydrase. The function of this family of proteins is to catalyze the reversible hydration of CO 2 , forming one bicarbonate ion and one proton, according to the following reaction: H 2 O + CO 2 -> H 2 CO 3 -> HCO 3

-+ H + . Since the first report by Miyamoto and co-workers of Nacrein -a modular protein with two carbonic anhydrase domains intercalated by an acidic domain -from the mollusk shell of the pearl oyster Pinctada fucata [18], many proteins in shells and other structures were identified by a one-per-one approach using classical molecular biology and biochemistry techniques. Until recently, the existing information was manageable and consisting of some proteins with very specific signatures: secretion signals, low complexity regions, repeats and some predictable domains (acidic, carbonic anhydrase, chitin-binding). The application of high-throughput techniques, genomics, transcriptomics and proteomics has changed the perspective, by revealing a much wider range of proteins that may be taking part in the control of biocalcification [19][20][21][22]. This clearly suggests broader functions for the OM proteins such as cross-communication between the mineral-producing tissue and the extracellular calcifying matrix, which go far beyond the catalysis of crystal nucleation and the guiding of mineral growth via binding to specific crystal surfaces. Here we present an overview of the proteins that have been identified in CaCO 3 -mineralized tissues of some metazoan groups currently available on UniprotKB [23,24]. We characterize this protein dataset from a functional viewpoint by using the Gene Ontology (GO) terms associated to the sequences [25].

Functional analysis of CaCO 3 -biomineralization proteins: data gathering

Keyword searches related to CaCO 3 -biomineralization processes were performed against the UniProtKB on March 2013 with taxonomic filters for metazoan non-vertebrate aquatic calcifying phyla: Porifera, Cnidaria, Brachiopoda, Mollusca, Echinodermata. This output was manually confirmed in order to have a representative set of reference proteins (Table 1). The most represented group is by far Mollusca due to their economical interest as food source and as 'providers' of cultured pearls. Consequently, these last years have seen an increased application of highthroughput approaches to molluscan tissues in combination with available genomes [26]. Because of the importance of reef-ecosystems in connection to environmental concerns at global scale -such as ocean acidification -, more sequences related to calcification have recently been identified from coral skeletons [19,22]. However the number of sequences available for these organisms is still low. Also for echinoderms the number of confirmed proteins remains limited [START_REF] Veis | Organic Matrix-related mineralization of sea urchin spicules, spines, test and teeth[END_REF]. Finally, sponges and brachiopods are not popular models in biomineralization studies, exhibiting the smallest publication rate in the field, particularly when regarding molecular characterizations of the organic matrix [28,29]. They consequently remain the groups with the lowest number of identified proteins involved in calcification. Each set of proteins was characterized for their content in GO terms belonging to the categories -Molecular Function (MF), Cellular Component (CC) and Biological Process (BP). We selected the top 5 GO terms of each group with more than one hit (Fig. 1 and Fig. 3). Biological Process (BP) terms were only included in mollusks (Fig. 2) as only this group of aquatic organisms had specific ontologies of the biomineralization process (shell calcification, GO:0031215). GO terms provide good predictions of protein function, and more broadly of the whole organic matrix, since a good share of the proteins listed in Table 1 are associated to ontologies. Fig. 1 summarizes the top GO terms, in specific categories, associated to shell matrix proteins in mollusks. The main protein functions relate to the catalytic activity of carbonic anhydrases (e.g. nacreins) and hydrolases, many types of binding functions like metal ion binding, carbohydrate and polysaccharide (e.g. perlucin, lustrin A) and enzyme regulatory activity (e.g. the proteins PSPI1 and NSPI1, containing protease inhibitor domains).

As discussed in earlier works [21,22,30], because the biomineral formation takes place extracellularly for each of the considered phylum, they are expected to have extracellular region and proteinaceous extracellular matrix as the main GO terms associated to protein localization. In corals and echinoderms, top molecular functions (MF, Fig. 3) are enzymatic and binding/structural. There is equivalent distribution of protein localization regarding the extracellular matrix environment and the cell membrane. This suggests a higher content of transmembrane proteins [22,31] in the OM of these organisms than in the OM of mollusks, for which the proportion of proteins targeted to the extracellular space is exceptionally high. These differences may reflect fundamental differences in the molecular process of biomineral formation in corals and echinoderms, on the one hand, and in molluscs on the other hand: while the first ones may involve both extracellular proteins and proteins with transmembrane domains -a fact that was evidenced for corals [22], extracellular proteins are the dominant constituents of shell matrices in mollusks. 3.

Conclusions

The analysis of gene ontology terms associated to biomineralization proteins brings an additional resource of information to characterize the organic matrix from a functional viewpoint. Indeed automatic annotations cover a significant number of the proteins considered in this study -50% in mollusks, 70% in corals and 83 % in echinoderms. it is important to alert those dealing with the discovery of novel proteins to the necessity of providing detailed information on the experimental characterization upon submission of the nucleotide and protein sequences to generalist databases (e.g. DDBJ/EMBL/GenBank, UniProtKB). Only with the help of the bench scientist, it is possible to ensure a faster update process and accurate information on sequence data immediately available to the community. Indeed, there are only a few biomineralization-specific ontologies, which include otolith and eggshell formation in vertebrates (e.g., otolith morphogenesis (GO:0032474), otolith mineralization (GO:0045299), eggshell formation (GO:0030703)), but also shell calcification in mollusks (GO:0031215) and chitin-based cuticle sclerotization in crustaceans (GO:0036340). In addition GO vocabularies specifically developed for CaCO 3 -biomineralization in other organisms are scarce. The community who studies organic matrix molecules can also contribute to the enrichment of the currently available vocabulary by suggesting new terms via the GO curators. These initiatives will greatly contribute to faster identification of biomineralization proteins in silico for non-model organisms and lead to better-automated annotations.

To conclude, the analysis of GO terms contributes to a general overview of the proteins associated to CaCO 3 -tissues. Fig. 4 summarizes the main types of molecules in terms of the primary structure, localization and possible role in calcification.

Fig. 1 -

 1 Fig. 1 -Top 5 (> 1 hit) Gene Ontology terms associated to shell matrix proteins (mollusks) within specific GO categories of Molecular Function (MF), and Cellular Component (CC).

Fig. 2 -

 2 Fig. 2 -Gene Ontology view of the Biological Processes (BP) associated to the shell matrix proteins dataset (mollusks).

Fig. 3 -

 3 Fig. 3 -Top 5 (> 1 hit) GO terms associated to skeletal matrix proteins (Cnidarians) and test, spine and tooth matrix proteins (Echinoderms). GO categories: Molecular Function (MF) and Cellular Component (CC).

Fig. 4 -

 4 Fig. 4 -Summary of the main types of biomineralization proteins in terms of the primary structure, localization and possible role in calcification.

Table 1 -

 1 Summary of the proteins identified as directly involved in metazoan CaCO 3biomineralization collected from the UniprotKB (March 2013). The final set was obtained based on keyword search and the following criteria: (1) Experimentally proved to be involved in biomineralization and (2) Inferred by homology (i.e. homologous counterparts in other organisms).

	Phylum	# total sequences	Calcified structure	Functions	# biomineralization proteins	# biomineralization proteins with GOs
	Mollusca	34370	Shell	Protection	355	178
	Echinodermata	28394	Test, Spine, Teeth	Protection	33	27
	Cnidaria	16132	Skeleton/Sclerites	Support	81	57
	Brachiopoda	135	Shell	Protection	2	0
	Porifera	26219	Spicules, Skeleton	Support	9	5

Acknowledgments

The work of P.R.-S. was supported by the Marie Curie ITN (Initial Training Network), BIOMINTEC piloted by the Johannes Gutenberg University, Mainz, Germany (Pr. H. C. Schröder, see www.biomintec.de) and the BioPreDyn project, coordinated by the Centre for Genomic Regulation (CRG) in Barcelona (see www. biopredyn.eu). Additional support included the COST Action TD0903 ('Biomineralix') during the 2012-2013 period. The work of F. Marin was also supported by an INTERRVIE project from the CNRS-INSU in the same period.