WO2026017834A1 - Inkjet-assisted enzymatic nucleic acid synthesis and cleavage - Google Patents

Abstract

Methods, devices, and compositions are provided for inkjet-assisted synthesis and recovery of a plurality of polynucleotides at reaction sites on a substrate comprising cleavable initiator nucleic acids, using template-free polymerases. Compositions include printable stabile formulations of cleavage reagents for inkjet delivery including, but not limited to, endonucleases, pH buffer and magnesium.

Description

INKJET-ASSISTED ENZYMATIC NUCLEIC ACID SYNTHESIS AND CLEAVAGE INTRODUCTION [001] Inkjet printing is a low-cost versatile technology for non-contact delivery of defined quantities of liquids to precise locations with minimal wastage. The technology has been applied to synthesis of oligonucleotide microarrays using phosphoramidite chemistry and has been employed to directly print enzymes onto substrates in the production of enzyme-based biosensors. In regard to the latter applications of inkjet printing, it has been observed that not only is enzyme activity affected by shear forces and the rheological requirements for droplet formation, but also by the changing enzyme concentration and buffer conditions from evaporative loss when, for example, enzyme-containing fluids are printed to microarrays, e.g. Di Risio et al, Macromolecular Rapid Comm., 28(18-19): (2007); Nishioka et al, J. Amer. Chem. Soc., 126(50): 16320-16321 (2004). [002] Recently there has been an interest in applying enzyme-based polynucleotide synthesis to problems which are ill-suited for conventional chemically based DNA synthesis, largely because of the mild aqueous reaction conditions of the enzymatic process. However, in addition to the above-mentioned difficulties of inkjet-delivery of enzymes, the use of enzymes presents a host of additional problems for any automated multi-step synthesis process including, enzyme adhesion to surfaces, the need for stringent temperature and pH control to maintain enzyme activity, aggregation of enzymes resulting in loss of activity and/or clogging of tubing, reaction sites or nozzles, variations in enzyme activity in or near synthesis supports, batch to batch differences in enzyme specific activity, the formation of foams or bubbles that inhibit reagent transfer and separation, loss of efficiency from reaction of certain protection groups with environmental contaminants, such as formaldehyde, and the like. [003] The ability to carry out inkjet reagent delivery for enzyme-based synthesis of dense arrays of polynucleotides would provide not only a convenient desk top synthesis method using aqueous reagents without the need for extensive environmental controls, but also significant advances in several diverse fields, including DNA data storage and cell and tissue analysis, such as, by direct labeling of viable biological cells, direct synthesis of spatial barcodes on tissues, and the like. [004] Significant progress has been made in the field. (Verardo et al., Sci Adc., 9, eadi0263, 2023). In certain cases, the recovery step is carried out by photocleaving. This requires introducing a UV-cleavable group in the initiator nucleic acid and contains the risk of introduction undesirable mutations during the UV-cleaving step. Alternatively, enzymatic bulk cleavage, wherein the polynucleotides are recovered in bulk after an incubation step with a specific nuclease has been proposed. This method leads to the obtention of mixed polynucleotides in a saline solution, which needs to be purified prior to subsequent downstream amplification steps such as PCR. In addition, such bulk recovery in solution does not allow for partial recovery of only a specific subset of polynucleotides from a microarray, in a customized manner. Similarly, the method of the prior art using a bulk solution does not allow the recovery of different amounts of different polynucleotides from a microarray, in a customized manner. [005] Thus, there remains a need in the art for a method for enzymatically cleaving a plurality of polynucleotides on a plurality of reaction sites on a substrate that is compatible with a plurality of downstream amplification steps and/or customizable. SUMMARY OF THE INVENTION [006] Methods, devices, and compositions are provided for inkjet-assisted cleavage of a plurality of polynucleotides at reaction sites on a substrate using endonuclease V. Compositions include printable stable formulations of cleavage reagents for inkjet delivery including, but not limited to, endonuclease V. [007] In one aspect, the invention relates to a method for enzymatically cleaving a plurality of polynucleotides or a subset of said plurality, each comprising a deoxyinosine on a substrate, wherein the substrate comprises a plurality of reaction sites and each polynucleotide of the plurality is assigned to a reaction site; the method comprising dispensing through one or more inkjet printhead nozzles at least one droplet of a printable reagent composition comprising an endonuclease V to each reaction site of the plurality or a subset of said reaction sites. [008] In another aspect, the invention relates to a method for enzymatically synthesizing a plurality of polynucleotides each having a predetermined sequence at reaction sites on a substrate, the method comprising: (a) providing the substrate, wherein the substrate comprises initiators at a plurality of reaction sites, wherein each initiator comprises a deoxyinosine penultimate to a 3’-terminal nucleotide having a free 3’-hydroxyl, and wherein each polynucleotide of the plurality is assigned to a reaction site for synthesis; (b) providing a set of printable reagent compositions each comprising a template-free polymerase and a 3’-O-protected nucleoside triphosphate; (c) performing a reaction cycle comprising the steps of i) dispensing through one or more inkjet printhead nozzles at least one droplet of one of the printable reagent compositions to each reaction site of the plurality where the 3’-O-protected nucleoside triphosphate is to be added, wherein the initiator or elongated fragments having free 3’-O-hydroxyls are reacted with the 3’-O-protected nucleoside triphosphate under suitable conditions for elongation by the template-free polymerase, wherein the initiator or elongated fragments are elongated by incorporation of the 3’-O-protected nucleoside triphosphate to form 3’-O-protected elongated fragments, and (ii) dispensing through one or more inkjet printhead nozzles at least one droplet of a deprotection solution to deprotect the 3’-O-protected elongated fragments to form elongated fragments having free 3’-hydroxyls; (d) repeating step (c) until the plurality of polynucleotides is synthesized; and (e) dispensing through one or more inkjet printhead nozzles at least one droplet of a printable reagent composition comprising an endonuclease V to each reaction site of the plurality or a subset thereof. [009] In another aspect, the invention relates to a printable reagent composition comprising - - an endonuclease V, preferably an endonuclease V selected from the group consisting of polypeptides having at least 80% identity with wild-type Endo V from E. coli (SEQ ID NO:1) and variants having at least 80% identity with the synthetic variant having the sequence set forth in SEQ ID NO:2, - a buffer, - a viscosity modifier, - a surface tension modifier, - MgCl_2, - a salt selected from NaCl and (NH4)2SO4, - and optionally BSA. BRIEF DESCRIPTION OF THE DRAWINGS [0010] FIG.1 shows a microscope image and schematics of a iDNA printed slide that was consecutively EndoV-cleaved via printing via a specific pattern. [0011] FIG. 2 shows the Typhoon image related to Figure 1 and the corresponding calculated cleaving yields. The first row represents experiments at 160 pulses of EndoV ink (top half of the slide in Figure 1) while the second row depicts experiments at 180 pulses of EndoV ink (lower half of the slide in Figure 1), wherein 100 pulses deliver 300pL. [0012] FIG. 3 shows the surface cleaving yield dependency on salt concentrations. [0013] FIG.4 shows the average Cq and dCq calculated based on qPCR assay for cleaving yield. [0014] FIG. 5 shows the relative activity of inks aged at 20°C for 1 or 2 weeks, compared to their fresh activity, as determined by the FRET assay in solution at 37°C. [0015] FIG. 6 shows the effect of BSA as a stabilizing agent on surface cleaving yield, PCR and aging. DETAILED DESCRIPTION [0016] Before the present methods, devices, and compositions are described, it is to be understood that this invention is not limited to particular methods, devices, or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0017] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction. [0019] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. [0020] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides, and reference to "the initiator" includes reference to one or more initiators and equivalents thereof, known to those skilled in the art, and so forth. [0021] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. Definitions [0022] The term "about", particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent. [0023] The terms “polynucleotide” “oligonucleotide”, “nucleic acid”, and “nucleic acid molecule” are used herein to include a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms “polynucleotide”, “oligonucleotide”, “nucleic acid”, and “nucleic acid molecule” include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oregon, as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. There is no intended distinction in length between the terms “polynucleotide”, “oligonucleotide”, “nucleic acid”, and “nucleic acid molecule,” and these terms will be used interchangeably. Thus, these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide. In particular, DNA is deoxyribonucleic acid. Monomers making up polynucleotides and oligonucleotides are capable of specifically binding to a natural polynucleotide by way of a regular pattern of monomer-to- monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Whenever the use of an oligonucleotide, polynucleotide, or nucleic acid requires enzymatic processing, such as extension by a polymerase, ligation by a ligase, or the like, one of ordinary skill would understand that oligonucleotides or polynucleotides in those instances would not contain certain analogs of internucleosidic linkages, sugar moieties, or bases at any or some positions. Polynucleotides typically range in size from a few monomeric units, e.g., 5-40, when they are usually referred to as “oligonucleotides,” to several thousand monomeric units. Whenever a polynucleotide, oligonucleotide, or nucleic acid is represented by a sequence of letters (upper or lower case), such as "ATGCCTG," it will be understood that the nucleotides are in 5'→3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, “I” denotes deoxyinosine, “U” denotes uridine, unless otherwise indicated or obvious from context. Unless otherwise noted the terminology and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Usually, polynucleotides comprise the four natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs, e.g., including modified bases, sugars, or internucleosidic linkages. It is clear to those skilled in the art that where an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g. single stranded DNA, RNA/DNA duplex, or the like, then selection of appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill, especially with guidance from treatises, such as Sambrook et al, Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989), and like references. Likewise, the oligonucleotide and polynucleotide may refer to either a single stranded form or a double stranded form (i.e. duplexes of an oligonucleotide or polynucleotide and its respective complement). It will be clear to one of ordinary skill which form or whether both forms are intended from the context of the term’s usage. [0024] The terms “base protecting moiety” and “base protecting group” are used interchangeably and refer to a protecting group on a nucleotide base, which may be used to reduce or eliminate the formation of secondary structure in the course of polynucleotide chain extensions and/or prevent deamination (see, e.g., International Patent Application Publication No. WO 2021/018921, herein incorporated by reference in its entirety). A base protecting group may be attached to the 6-nitrogen of deoxyadenosine triphosphate, the 2-nitrogen of deoxyguanosine triphosphate, and/or the 4-nitrogen of deoxycytidine triphosphate. In some embodiments, a base protecting group is attached to all of the indicated nitrogens. In some embodiments, a base protecting group attached to a 6-nitrogen of deoxyadenosine triphosphate is selected from the group consisting of benzoyl, phthaloyl, phenoxyacetyl, and methoxy acetyl; a base protecting group attached to the 2-nitrogen of deoxyguanosine triphosphate is selected from the group consisting of isobutyryl, isobutyryloxyethylene, acetyl, 4-isopropyl- phenoxyacetyl, phenoxyacetyl, and methoxyacetyl; and a base protecting group attached to said 4-nitrogen of deoxycytidine triphosphate is selected from the group consisting of benzoyl, phthaloyl, acetyl, and isobutyryl. In some embodiments, a protecting group attached to the 6- nitrogen of deoxyadenosine triphosphate is benzoyl; a base protecting group attached to the 2- nitrogen of deoxyguanosine triphosphate is isobutryl or dimethylformamidine; and the base protecting group attached to the 4-nitrogen of deoxycytidine triphosphate is acetyl. In some embodiments, a base protecting group attached to the 6-nitrogen of deoxyadenosine triphosphate is phenoxyacetyl; a base protecting group attached to the 2-nitrogen of deoxyguanosine triphosphate is 4-isopropyl-phenoxyacetyl or dimethylformamidine; and the base protecting group attached to the 4-nitrogen of deoxycytidine triphosphate is acetyl. In some embodiments, base protecting moieties are removed (i.e. the product is deprotected) and product is cleaved from a solid support in the same reaction. For example, an initiator may comprise a ribo-uridine which may be cleaved to release the polynucleotide product by treatment with 1 M KOH, or like reagent (ammonia, ammonium hydroxide, NaOH, or the like), which simultaneously removes base-labile base protecting moieties. [0025] As used herein, the term “initiator”, “DNA initiator”, “initiating fragment”, “initiator nucleic acid”, “initiator oligonucleotide”, or “initiator polynucleotide” refers to an oligonucleotide or polynucleotide comprising a free 3’-hydroxyl group, which can be further elongated by a template-free polymerase (e.g., TdT). In one embodiment, the initiator is a DNA initiating fragment. In an alternative embodiment, the initiator is an RNA initiating fragment. In some embodiments, an initiator comprises between 3 and 100 nucleotides. In some embodiments, an initiator comprises between 3 and 20 nucleotides. In some embodiments, the initiator is single-stranded. In alternative embodiments, the initiator is double- stranded. In some embodiments, an initiator may comprise a non-nucleic acid compound having a free hydroxyl group to which a TdT may couple a 3’-O-protected dNTP (see, e.g., Baiga, U.S. patent publications US2019/0078065 and US2019/0078126; herein incorporated by reference). [0026] According to the present invention, the initiator comprises a deoxyinosine penultimate to a 3’-terminal nucleotide having a free 3’-hydroxyl. [0027] As used herein, the term "extension product", “extension intermediate”, “elongation product” or “elongation intermediate” are used interchangeably and refer to the product resulting from enzymatic extension at the 3' end of an initiator or of an extension intermediate generated from an initiator by a template-free polymerase. [0028] As used herein, “inkjet assisted synthesis” means that one or more synthesis reagents are delivered to reaction sites in droplets generated by one or more inkjet printhead nozzles. [0029] “Synthesis reagents” include any reagent used in a synthesis cycle to couple a nucleotide monomer, particularly a 3’-O-protected-nucleoside triphosphate, to an initiator or elongated fragment. Synthesis reagents may include a template-free polymerase, cofactors (e.g., Co²⁺ or other divalent cations), and nucleotides (e.g., 3’-O-protected-nucleotides), buffers, deprotection or deblocking agents, and the like. [0030] “Synthesis reagents” also include reagents for preparing a substrate for polynucleotide synthesis, such as, reagents for defining reaction sites, initiators, capping reagents, and the like. [0031] The terms “deprotection” agent, deprotection buffer, deprotection solution, and the terms “deblocking” agent, deblocking buffer, and deblocking solution are used herein interchangeably. Likewise, the term “protected” in reference to compounds, such as, dNTPs, is used synonymously with the term “blocked” in reference to compounds. [0032] As used herein, the term “deprotection solution” (or its equivalent terms) means a reagent that brings about or promotes the removal of a protection group, for example, a 3’-O- protecting group of a nucleotide. As described more fully below, the composition of a deprotection solution (and deprotection reaction conditions) depends on the nature of the protecting group (or blocking group) which is to be removed. In various embodiments, a deprotection solution may contain specific reagents that chemically react with a protection group and/or protected moiety (such as, a reducing agent like tris(2-carboxyethyl)phosphine (TCEP), enzymes for enzymatic cleavage, scavengers, co-factors, or the like). In some embodiments, a deprotection solution may not contain specific reagents that react with a protection group, but may contain components, e.g., pH buffers, that are compatible with or promote physical cleavage of a protecting group, such as in the case of a photocleavable protecting group. Typically, in a reaction cycle for elongating a polynucleotide fragment, in a deprotecting step a deprotection solution is incubated with 3’-O-protected elongated fragments for a predetermined incubation time. Typical incubation times for the deprotection step (i.e. durations of the deprotection step) are in the range of from 1 minute to 15 minutes, or in the range of from 1 minute to 10 minutes, preferably from 2 to 5 minutes, such as about 3 minutes. [0033] For the elongation step, typical incubation times (i.e. durations of elongation steps) are in the range of from 1 minute to 30 minutes; or in the range of from 3 minutes to 30 minutes; or in the range of from 3 minutes to 15 minutes. Typical elongation reaction temperatures are in the range of from room temperature (RT) to 80^oC; or from 20^oC to 80^oC; or from 20^oC to 60^oC. [0034] As used herein, the term “cleaving formulation” or “cleaving ink composition” (or its equivalent terms) means a composition that comprises an endonuclease V in a suitable solution. [0035] As used herein, the terms “Endonuclease V” or “Endo V” are used interchangeably and mean any enzyme that possesses deoxyinosine 3’ endonuclease activity, i.e. the ability to recognize DNA containing deoxyinosine (a deamination product of a deoxyadenosine, also referred as inosine and hypoxanthine) residues. Endo V primarily cleaves the second phosphodiester bond 3’ to an inosine residue in the same strand, leaving a nick with a 3’- hydroxyl and a 5’-phosphate. [0036] The most widely used Endo V is the wild-type Endonuclease V from E. coli. (having the sequence set forth in SEQ ID NO:1), such as the E coli Endo V commercialized by NEB under catalog number M0305S. Variants of E coli Endo V have also been described, that exhibit improved stability and/or activity (e.g. Loftie-Eaton et al. International patent publication WO2022/090057). Synthetic Endo V polypeptides have also been developed, such as the polypeptide having the sequence set forth in SEQ ID NO:2. [0037] A “distinct reaction site” on a substrate is a discrete site in that it is separated from other reaction sites; that is, a discrete site does not have a border with, or overlap with, another reaction site. In other words, a discrete or different reaction site is not contiguous with, or overlapping with, other reaction sites. Exceptions to this usual arrangement include “overwriting” embodiments described below for generating high density barcodes on surfaces. [0038] A solid support is “addressable” when it has multiple features (e.g., reaction centers) positioned at particular predetermined locations (e.g., “addresses”) on the surface of the solid support. [0039] An “array” includes any two-dimensional or substantially two-dimensional (as well as a three-dimensional) arrangement of addressable regions, e.g., spatially addressable regions. An array is “addressable” when it has multiple features (e.g., reaction centers) positioned at particular predetermined locations (e.g., “addresses”) on the array. Array features may be separated by intervening spaces. [0040] “Primer” means an oligonucleotide, either natural or synthetic that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3’ end along the template so that an extended duplex is formed. Extension of a primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase. The sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide. Usually, primers are extended by a DNA polymerase. Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers. Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skill in the art, as evidenced by the following references that are incorporated by reference: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, New York, 2003). [0041] "Sequence determination", “sequencing” or "determining a nucleotide sequence" in reference to polynucleotides includes determination of partial as well as full sequence information of the polynucleotide. That is, the terms include sequences of subsets of the full set of four natural nucleotides, A, C, G and T, such as, for example, a sequence of just A’s and C’s of a target polynucleotide. That is, the terms include the determination of the identities, ordering, and locations of one, two, three or all of the four types of nucleotides within a target polynucleotide. In some embodiments, the terms include the determination of the identities, ordering, and locations of two, three or all of the four types of nucleotides within a target polynucleotide. In some embodiments sequence determination may be accomplished by identifying the ordering and locations of a single type of nucleotide, e.g. cytosines, within the target polynucleotide "catcgc . . . " so that its sequence is represented as a binary code, e.g., "100101... " representing "c-(not c)(not c)c-(not c)-c ... " and the like. In some embodiments, the terms may also include subsequences of a target polynucleotide that serve as a fingerprint for the target polynucleotide; that is, subsequences that uniquely identify a target polynucleotide within a set of polynucleotides, e.g. all different RNA sequences expressed by a cell. [0042] “Substantially purified” generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically, in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90- 95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density. [0043] By “isolated” is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type. The term “isolated” with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome. [0044] “Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80%-85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence. [0045] As used herein, the term “sequence identity” or “identity” refers to the number (or fraction expressed as a percentage %) of matches (identical amino acid residues) between two polypeptide sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http://blast.ncbi.nlm.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, % amino acid sequence identity values refers to values generated using the pair wise sequence alignment program EMBOSS Needle that creates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm, wherein all search parameters are set to default values, i.e. Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5. [0046] The terms "modification" or "alteration" as used herein in relation to a position or amino acid mean that the amino acid in the specific position has been modified compared to the amino acid of the wild-type protein. [0047] A "substitution" means that an amino acid residue is replaced by another amino acid residue. For example, the term "substitution" refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g. hydroxyproline, hydroxylysine, allohydroxylysine, 6-N- methylysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N- methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and non-naturally occurring amino acid residue, often made synthetically, (e.g. cyclohexyl-alanine). [0048] Amino acids may be represented by their one-letter or three-letters code according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (Ile); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y: tyrosine (Tyr). [0049] A substitution can be a conservative or non-conservative substitution. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine and serine). [0050] Herein, the terms "peptide", "polypeptide", "protein", "enzyme", refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain. [0051] “Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions. [0052] The terms "connected" or "coupled" are used in an operational sense and are not necessarily limited to a direct connection or coupling. For example, two devices or components may be coupled directly, or via one or more intermediary media or devices. As another example, devices may be coupled in such a way that information or data can be passed between them, while not sharing any physical connection with one another. In some cases, two devices or components may be connected by a wire or wirelessly to each other. Inkjet-Assisted Enzymatic Synthesis and Recovery of Polynucleotides [0053] Methods and compositions are provided for inkjet-assisted synthesis of a plurality of polynucleotides and recovery of said plurality of polynucleotides or a subset thereof, each at a distinct reaction site on a substrate, using a template-free polymerase and an endonuclease V. Typically, enzymatic synthesis of polynucleotides takes place on substrates comprising a planar surface, such as, glass, silica, silicon oxide, plastic, or like surfaces, but it may also take place on other surfaces, such as, for example, non-planar surfaces (e.g., beads, particles), biological tissues, or surface-immobilized cDNAs extracted from tissues. The methods disclosed herein may include the use of an inkjet printer for highly parallel template-free enzymatic synthesis of polynucleotides, as described in International Application Publication No. WO 2022/013094; herein incorporated by reference in its entirety. The plurality of polynucleotides or a subset thereof are then recovered from the plurality of reactions sites after a cleaving step using an endonuclease V. The method of the invention also allows the recovery of polynucleotides from only a subset of the reaction sites, for example at a given point in time, more polynucleotides being recovered later in time. It also allows the recovery of polynucleotides from different subsets of reaction sites in different amounts. [0054] In one aspect, the invention relates to a method for enzymatically cleaving a plurality of polynucleotides or a subset thereof, each comprising a deoxyinosine on a substrate, wherein the substrate comprises a plurality of reaction sites and each polynucleotide of the plurality is assigned to a reaction site; the method comprising dispensing through one or more inkjet printhead nozzles at least one droplet of a printable reagent composition comprising an endonuclease V to each reaction site of the plurality or to a subset of said reaction sites. [0055] In another aspect, the invention relates to a method for enzymatically synthesizing a plurality of polynucleotides each having a predetermined sequence at reaction sites on a substrate, the method comprising: (a) providing the substrate, wherein the substrate comprises initiators at a plurality of reaction sites, wherein each initiator comprises a deoxyinosine penultimate to a 3’-terminal nucleotide having a free 3’-hydroxyl, and wherein each polynucleotide of the plurality is assigned to a reaction site for synthesis; (b) providing a set of printable reagent compositions comprising a template-free polymerase and a 3’-O-protected nucleoside triphosphate, (c) performing a reaction cycle comprising the steps of i) dispensing through one or more inkjet printhead nozzles at least one droplet of one of the printable reagent compositions to each reaction site of the plurality where the 3’-O-protected nucleoside triphosphate is to be added, wherein the initiator or elongated fragments having free 3’-O-hydroxyls are reacted with the 3’-O-protected nucleoside triphosphate under suitable conditions for elongation by the template-free polymerase, wherein the initiator or elongated fragments are elongated by incorporation of the 3’-O-protected nucleoside triphosphate to form 3’-O-protected elongated fragments, and (ii) dispensing through one or more inkjet printhead nozzles at least one droplet of a deprotection solution to deprotect the 3’-O-protected elongated fragments to form elongated fragments having free 3’-hydroxyls; (d) repeating step (c) until the plurality of polynucleotides is synthesized; and (e) dispensing through one or more inkjet printhead nozzles at least one droplet of a printable reagent composition comprising an endonuclease V to each reaction site of the plurality or a subset thereof. [0056] In some embodiments, the plurality of polynucleotides may be in the range of from 2 to 500,000; or in the range of from 100 to 400,000; or in the range of from 100 to 200,000; or in the range of from 100 to 100,000. The plurality of polynucleotides may be the same or different than the plurality of reaction sites. In some embodiments, the plurality of reaction sites may be greater than the plurality of polynucleotides. In some embodiments, the above pluralities of reaction sites each have a density equivalent to that if uniformly deposited on an area equivalent to that of a standard 25 mm x 75 mm microscope slide. In some embodiments, an array of reaction sites formed by uniform deposition may be a rectilinear array; and in other embodiments, an array of reaction sites formed by uniform deposition may be a hexagonal array. [0057] In some embodiments, parallel synthesis is implemented by providing a support having discrete, non-overlapping, addressable sites where separate polynucleotides are synthesized and a means for controlling photoillumination, electrochemical conditions, or other reaction conditions at each site independently of the other sites. In some embodiments, such a parallel synthesis support is a planar support having a regular pattern of addressable sites, such as, a rectilinear pattern of sites, or a hexagonal pattern of sites. In some embodiments, the support is a planar support having an irregular pattern of addressable sites or complex pattern of addressable sites. In some embodiments, each site of a planar support is associated with one or more electrodes whose electrical characteristics may be controlled in an addressable manner independent of other electrodes of the planar support. [0058] In some embodiments, the planar support comprises a plurality of sites comprising at least 256 sites, at least 512 sites, at least 1024 sites, at least 5000 sites, at least 10,000 sites, at least 25,000 sites, or at least 100,000 sites and as many as 10,000,000 sites. In some embodiments, such planar supports have a plurality of sites greater than 1000, or 10,000, or 25,000, or 50,000, or 100,000, or 500,000, and up to 1,000,000 sites or up to 10,000,000 sites, or up to 300,000,000 sites. In some embodiments, the sites of the planar support are disposed in a regular array and each site is associated with at least one electrode integrated with the planar support. In some embodiments, the number of polynucleotides synthesized at each site is in the range of from 1000 molecules to 10⁶ molecules, or from 1000 molecules to 10⁹ molecules, or from 1000 molecules to 10¹² molecules. [0059] In some embodiments, enzymatically synthesized polynucleotides at each reaction site have lengths in the range of from 50 to 1000 nucleotides. [0060] Template-free enzymatic synthesis of polynucleotides involves cycles of steps with most involving delivery to a reaction site of at least one of the following reagents: a composition comprising a template-free polymerase, a composition comprising any cofactors needed for activity of the template-free polymerase, one or more compositions, each comprising one or more 3’-O-protected-dNTPs (i.e., monomers), a composition comprising reagents for deprotection, a composition comprising cleaving reagents such as Endonuclease V and wash solutions. In various embodiments, the compositions comprising the template-free polymerase, cofactors, 3’-O-protected-dNTPs, reagents for deprotection, endonuclease V and wash solutions may be conveyed to reaction sites by droplets created and delivered by inkjet printhead nozzles. To be delivered by inkjet-generated droplets, these reagents must be formulated to meet the rheological requirements for droplet formation. These formulations are referred to as “inks”. The key rheological parameters affecting droplet formation are viscosity, density and surface tension, e.g. Derby, Annu. Rev. Mater. Sci., 40: 395-414 (2010); Derby, J. Mater. Chem., 18: 5717-57-21 (2008); Calvert, Chem. Mater., 13: 3299-3305 (2001); Tekin et al, Soft Matter, 4: 703-713 (2008); and like references. Another key parameter relating to droplet volume is the nozzle diameters of the inkjet printhead nozzles. In some embodiments, nozzle diameters may be in the range of from 10 µm to 100 µm, including any diameter within this. [0061] In one aspect, reagent inks are provided for inkjet-assisted enzymatic cleavage of polynucleotides, and in particular, inks comprising endonuclease V, as described further below. Template-Free Enzymatic Synthesis of Polynucleotides [0062] Enzymatic nucleic acid synthesis uses an enzymatic catalyst to carry out the polymerization of nucleotides. Enzymatic DNA synthesis is generally performed with an enzyme that catalyzes the addition of nucleotides to the 3' end of a DNA molecule. More specifically, the process employs, without being limited thereto, enzymes which make possible the creation of a phosphodiester bond between a 3'-OH group of a nucleic acid fragment in the course of synthesis and the 5'-OH group of the nucleotide to be added during the enzymatic addition stage. [0063] In some embodiments, enzymatic nucleic acid synthesis is performed with an enzyme capable of catalyzing the polymerization of nucleotides independently of the presence of a complementary strand (i.e., template). Such enzymes are capable of synthesizing nucleic acids in the absence of any complementary strand. In some cases, enzymatic nucleic acid synthesis may be performed with an enzyme that has the ability to synthesize single stranded nucleic acid fragments. The addition of nucleotides is thus advantageously carried out by the enzymatic route, by means of enzymes capable of polymerizing nucleotides without the presence of a template strand. [0064] In some embodiments, the enzyme chosen for use in enzymatic nucleic acid synthesis is a template-free polymerase selected from translesion DNA polymerases of type η (eta) or ζ (zeta), polynucleotide phosphorylases (PNPases), template-independent RNA polymerases, terminal transferases, template-independent DNA polymerases, reverse transcriptases, 9°N DNA polymerases, or terminal deoxynucleotidyl transferases (TdT). These enzymes are expressed by certain cells of living organisms and can be extracted from these cells or purified from recombinant cultures. [0065] In some embodiments, an engineered terminal deoxynucleotidyl transferase is used to perform enzymatic nucleic acid synthesis. Various variants of terminal deoxynucleotidyl transferase have been developed for this purpose. See, e.g., international patent applications published as WO 2020/099451; WO2021/116270; WO2021/213903, WO2022/063835; WO2023083997; WO2020/239737 and WO2023/083999.; herein incorporated by reference in their entireties. In some embodiments, an engineered reverse transcriptase is used to perform enzymatic nucleic acid synthesis. For example, human immunodeficiency virus type-1 and Moloney murine leukemia virus reverse transcriptases may be used. Engineered Moloney murine leukemia virus reverse transcriptase variants are commercially available such as the SuperScript IV reverse transcriptase from Thermo Fisher (Waltham, MA) and SMARTScribe reverse transcriptase from Clonetech (Mountain View, Calif.). [0066] In some embodiments, an engineered 9°N DNA polymerase is used to perform enzymatic DNA synthesis. Engineered 9°N DNA polymerase variants are commercially available, such as the Therminator Thermococcus sp. DNA polymerase from New England Biolabs (Ipswich, MA). See also, e.g., Hoff et al. (2020) ACS Synth Biol 9(2):283-293; Gardner et al. (2019) Front. Mol. Biosci. 6:28; herein incorporated by reference in their entireties. [0067] A cycle of the enzymatic synthesis process, leading to the addition of a nucleotide to a nucleic acid strand, comprises two successive steps, an elongation step and a deprotecting step respectively. During the elongation step, the polymerase adds a nucleotide comprising a protecting group to a nucleic acid strand. Then the protection group is removed from this newly added nucleotide, to be able to perform additional cycles. [0068] Synthesis of a complete nucleic acid by template-free enzymatic nucleic acid synthesis typically comprises repeated cycles of steps, in which a selected nucleotide is coupled to an initiator or growing chain in each cycle. [0069] The general elements of template-free enzymatic synthesis are described in the following references: Ybert et al, International patent publication WO/2015/ 159023; Ybert et al, International patent publication WO/2017/216472; Hyman, U.S. patent 5436143; Hiatt et al, U.S. patent 5763594; Jensen et al, Biochemistry, 57: 1821-1832 (2018); Mathews et al, Organic & Biomolecular Chemistry, DOI: 0.l039/c6ob0l37lf (2016); Schmitz et al, Organic Lett., 1(11): 1729-1731 (1999). [0070] In some embodiments, an initiator oligonucleotide may be attached to a synthesis support by its 5’end; and in other embodiments, an initiator oligonucleotide may be attached indirectly to a synthesis support by forming a duplex with a complementary oligonucleotide that is directly attached to the synthesis support, e.g. through a covalent bond. In some embodiments a synthesis support is a solid support which may be a discrete region of a solid planar solid, or may be a bead. [0071] According to the present invention, the initiator comprises a deoxyinosine penultimate to a 3’-terminal nucleotide having a free 3’-hydroxyl. This deoxyinosine residue is recognized by the Endonuclease V enzyme, which allows for the selective cleavage of the second phosphodiester bond 3’ to the deoxyinosine, thereby releasing the polynucleotides after synthesis. [0072] Initiators are provided, for example, attached to a solid support, with a free 3’- hydroxyl groups. To the initiator (or elongated initiator polynucleotides in subsequent cycles) are added a 3’-O-protected-dNTP and a template-free polymerase, such as a TdT or a variant thereof (e.g., Ybert et al, WO/2017/216472) under conditions effective for the enzymatic incorporation of the 3’-O-protected-dNTP onto the 3’-end of the initiator (or elongated initiator polynucleotides). This reaction produces elongated initiator polynucleotides whose 3’- hydroxyls are protected. If the elongated initiator polynucleotide does not contain a completed sequence, then the 3’-O-protection groups are removed to expose free 3’-hydroxyls and the elongated initiator polynucleotides are subjected to another cycle of nucleotide addition and deprotection. If the elongated initiator polynucleotide contains a competed sequence, then the 3’-O-protection group may be removed, or deprotected, and the desired sequence may be cleaved from the original initiator. Such cleavage may be carried out using any of a variety of single strand cleavage techniques, for example, by inserting a cleavable nucleotide at a predetermined location within the original initiator. An exemplary cleavable nucleotide may be a uracil nucleotide which is cleaved by uracil DNA glycosylase. In some embodiments, 3’- O-protection groups are electrochemically labile groups. That is, deprotection or cleavage of the protection group is accomplished by changing the electrochemical conditions in the vicinity of the protection group which result in cleavage. Such changes in electrochemical conditions may be brought about by changing or applying a physical quantity, such as a voltage difference or light to activate auxiliary species which, in turn, cause changes in the electrochemical conditions at the site of the protection group, such as an increase or decrease in pH. In some embodiments, electrochemically labile groups include, for example, pH-sensitive protection groups that are cleaved whenever the pH is changed to a predetermined value. In other embodiments, electrochemically labile groups include protecting groups which are cleaved directly whenever reducing or oxidizing conditions are changed, for example, by increasing or decreasing a voltage difference at the site of the protection group. [0073] In some embodiments, an ordered sequence of nucleotides is coupled to an initiator using a template-free polymerase, such as TdT, in the presence of 3’-O-protected dNTPs at each synthesis step. In some embodiments, the method of synthesizing an oligonucleotide comprises the steps of (a) providing an initiator having a free 3’-hydroxyl; (b) reacting under extension conditions the initiator or an extension intermediate having a free 3’-hydroxyl with a template-free polymerase in the presence of a 3’-O-protected nucleoside triphosphate to produce a 3’-O-protected extension intermediate; (c) deprotecting the extension intermediate to produce an extension intermediate with a free 3’-hydroxyl group; and (d) repeating steps (b) and (c) until the polynucleotide is synthesized. In some embodiments, an initiator is provided as an oligonucleotide attached to a solid support, e.g., by its 5’ end. The above method may also include washing steps after the reaction, or extension, step, as well as after the de- protecting step. For example, the step of reacting may include a sub-step of removing unincorporated nucleoside triphosphates, e.g., by washing, after a predetermined incubation period, or reaction time. Such predetermined incubation periods or reaction times may be a few seconds, e.g., 30 seconds, to several minutes, e.g., 30 minutes. [0074] The 3’-O-blocked dNTPs employed may be purchased from commercial vendors or synthesized using published techniques (see, e.g., U.S. Patent No.7,057,026; Guo et al, Proc. Natl. Acad. Sci., 105(27): 9145-9150 (2008); Benner, U.S. Patent No. 7,544,794; herein incorporated by reference in their entireties). [0075] The above method may also include capping step(s) as well as washing steps after the reacting, or extending, step, as well as after the deprotecting step. As mentioned above, in some embodiments, capping steps may be included in which non-extended free 3’-hydroxyl groups are reacted with compounds that prevents any further extension of the capped strand. In some embodiments, the compound is a dideoxynucleoside triphosphate. In other embodiments, non-extended strands with free 3’-hydroxyl groups are degraded by treating them with a 3’-exonuclease activity, e.g., Exo I. For example, see Hyman, U.S. Patent No. 5,436,143. Likewise, in some embodiments, strands that fail to be deblocked may be treated to either remove the strand or render it inert to further extensions. [0076] The methods according to the present invention comprise dispensing through one or more inkjet printhead nozzles at least one droplet of a printable reagent composition comprising an endonuclease V to each reaction site of the plurality or a subset thereof. [0077] The cleaving step is carried out under suitable conditions for the endonuclease V to release the synthesized polynucleotides. For the cleavage step, typical incubation times (i.e. duration of the cleavage step) are in the range of from 10 minutes to 24 hours; or in the range of from 30 minutes to 20h; or in the range of from 1 to 5 hours. Typical cleavage reaction temperatures are in the range of from room temperature (RT) to 80^oC; or from 20^oC to 80^oC; or from 20^oC to 50^oC, typically around 20°C, 37°C or 45°C. The skilled person will readily select the appropriate duration of the cleavage step, depending on the selection temperature, amount of enzyme etc. [0078] In certain embodiments, once the printable reagent composition comprising an endonuclease V has been dispensed, it is incubated for 1 hour at 37°C or 45°C, or for 20h at [0079] One of the advantages of the method of the present invention is that is allows the obtained polynucleotides to be used directly in subsequent nucleic acid amplification steps, without the need for desalting and/or purification steps. [0080] Therefore, in one embodiment, the plurality of polynucleotides obtained by said method can be used in a subsequent nucleic acid amplification step without the need for a desalting step prior to said amplification step. [0081] In one aspect, the present invention relates to a method for carrying out a nucleic acid amplification, wherein said method comprises the use of one or several polynucleotides obtained as described above and wherein said method does not comprise a purification step. Alternatively, the method for carrying out nucleic acid amplification can comprise a purification step. Any suitable method for purifying polynucleotides can be used. Typically, the purification step (also known as “desalting step”) can be carried out using a suitable filter, such as 3kDa filter (eg from Amicon®). Inks for Inkjet-Assisted Enzymatic Cleavage of Polynucleotides [0082] Reagents delivered by inkjet printhead nozzles must be formulated so as to preserve activity of reagents, avoid formation of precipitates that clog the printhead nozzle, and meet the rheological requirements for droplet formation. Printable reagent compositions are referred to herein as “inks.” As used herein, the term “printable reagent composition” or “ink” refers to a composition comprising a reagent that can be delivered through nozzles. It should therefore meet certain viscosity requirements, whilst preserving the activity of the reagent. The printing reagent compositions comprising an endonuclease V are also referred to as “cleavage ink” or “cleaving ink” or “EVi”. Also described herein are printable reagent compositions comprising a template-free polymerase (also termed “elongation inks”). [0083] For example, satisfying the first constraint (activity) may require that an endonuclease V be present in a reaction mixture at a certain minimal concentration. However, because of high protein viscosity, the concentration for the desired activity may interfere with the second constraint, i.e., that the reagent composition be capable of droplet formation. In some embodiments the endonuclease V may be delivered in a plurality of droplets, each with lower concentrations of endonuclease which coupled with evaporation permit the build-up of the endonuclease concentration to provide a desired level of activity. [0084] In some embodiments, inks comprise combinations of premixed synthesis reagents. Preferred combinations of synthesis reagents retain enzymatic activity without formation of precipitates under operating conditions for inkjet-assisted cleavage and remain stable during storage. In some embodiments, an ink is stable (i.e., retains enzymatic activity without formation of detectable precipitates) for at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 20 days, or at least 30 days, or longer. [0085] In some embodiments, a printable reagent composition is provided, the printable reagent composition comprising: - an endonuclease V, preferably an endonuclease V selected from the group consisting of polypeptides having at least 80% identity with wild-type Endo V from E. coli (SEQ ID NO:1) and variants having at least 80% identity with the synthetic variant having the sequence as set forth in SEQ ID NO:2, - a buffer, - a viscosity modifier, - a surface tension modifier, - MgCl2, - a salt selected from NaCl and (NH4)2SO4 - and optionally BSA. [0086] Any endonuclease V polypeptide displaying a deoxyinosine-specific nucleic acid cleavage activity can be used in the present invention. In some embodiments, the endonuclease V is selected from the group consisting of polypeptides having at least 80% identity with wild-type Endo V from E. coli (SEQ ID NO:1) and variants having at least 80% identity with the synthetic variant having the sequence set forth in SEQ ID NO:2. [0087] In some embodiments, the endonuclease V is a polypeptide having at least 80% identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with wild-type Endo V from E. coli (SEQ ID NO:1). [0088] In some embodiments, the endonuclease V is a polypeptide having at least 80% identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with synthetic variant having the sequence set forth in SEQ ID NO:2. [0089] In some embodiments, the endonuclease can contain a tag, which allows easy purification of said endonuclease. [0090] In some embodiments, the endonuclease V can be present in the printable reagent composition at a concentration of 0.5µM to 1 µM, preferably between 0.75 and 0.80 µM, even more preferably around 0.78µM. [0091] Reagent compositions may be filtered through one or more filters prior to dispensing to the reaction sites. To avoid adsorption of the template-free polymerase to the filter, preferably a low protein binding filter is used that does not absorb the enzyme or reduce enzymatic activity. Exemplary low protein binding filters include but are not limited to, polytetrafluoroethylene (PTFE) filters, cellulose acetate filters, glass fiber filters, polyethersulfone (PES) filters, polypropylene (PP) filters, polyvinylidene fluoride (PVDF) filters, regenerated cellulose (RC) filters, and Anopore™ inorganic membrane filters. [0092] In some embodiments, the printable reagent composition is filtered through at least one filter having a pore size ranging from 0.6 µm to 5 µm in diameter, including any pore size within this range. In some embodiments, the printable reagent composition is filtered through at least one filter, at least two filters, at least three filters, or more. The filters may have the same size or different sizes. In some embodiments, the printable reagent composition is filtered at least one time, at least two times, at least three times, or at least four times, or more. In some embodiments, the printable reagent composition comprising endonuclease V is filtered through at least one filter having a pore size less than or equal to 0.8 µm in diameter. [0093] In some embodiments, some reagents are prefiltered before mixing with other reagents to produce the printable reagent composition. In an exemplary embodiment, the printable reagent composition is prepared by a method comprising: mixing the divalent cation, the polar organic solvent, and the buffer to form a solution; filtering the solution through a first filter to form a filtered solution; adding the endonuclease V to the filtered solution to form the final printable reagent composition; and filtering the printable reagent composition through a second filter before dispensing. In some embodiments, the first filter has a pore size of 0.8 µm in diameter, and the second filter has a pore size of 5 µm in diameter. [0094] In some embodiments, printable reagent compositions such as the printable reagent composition comprising the template-free polymerase and a 3’-protected nucleoside triphosphate (elongation ink) are delivered in droplets ranging in volume from 1 pL to 200 pL, or from 1 pL to 100 pL, or from 1 pL to 50 pL, or preferably from 1 pL to 30 pL. Any volume in these ranges can be delivered in droplets as well. [0095] According to the present invention, the printable reagent composition comprising an endonuclease V (cleaving ink) is delivered in droplets ranging in volume from 100 pL to 1000 pL, or from 400 pL to 800 pL, or preferably from 450 to 650 pL. Any volume in these ranges can be delivered in droplets as well. [0096] In some embodiments, a printable reagent composition is dispensed by an ink jet at a temperature above 18 °C. For example, printable reagent compositions may be dispensed by an inkjet printer at a temperature ranging from 19 °C to 45 °C, including any temperature within this range. In some embodiments, the dispensing of a printable reagent composition is performed at room temperature. [0097] In some cases, reagent compositions (including, but not limited to, the printable reagent composition comprising a template-free polymerase and a 3’-protected nucleoside triphosphate and the printable reagent composition comprising an endonuclease V) may be stored before use in inkjet-assisted synthesis. In some embodiments, printable reagent compositions are stored for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer before use in inkjet-assisted synthesis. Reagent compositions may be stored, for example, at temperatures ranging from about -20 °C to room temperature. In some embodiments, the printable reagent composition is stored at a temperature in a range from -20 °C to 4 °C prior to said dispensing, including any temperature within this range. [0098] In some embodiments, the printable reagent composition comprising endonuclease V is stored at a temperature in a range from -20°C to 4°C for up to one month prior to dispensing. [0099] In some embodiments, printable reagent compositions are made shortly before performing inkjet-assisted cleavage. [00100] As also mentioned above, the key solution parameters affecting droplet formation by inkjets are viscosity, surface tension, density and diameter of the inkjet nozzle, which are related through the formula: Z=[(ργa)^(0.5)]/η, where ρ is the density of the fluid, γ is surface tension, η is viscosity, a is the radius of the inkjet printhead nozzle and Z is in the range of from 1 to 10 for reliable droplet formation, e.g. Derby, J. Mater. Chem., 18: 5717-5721 (2008). This relationship applies to any of the printable reagent compositions, described herein, that are delivered by inkjet-generated droplets. Applying this relationship to determine ink compositions that are capable of forming desired droplets for particular embodiments may be carried out by one of ordinary skill in the art by adjusting densities of reactants, viscosity modifiers, surface tension modifiers, and the like. [00101] Thus, in some embodiments, printable reagent compositions further comprise viscosity modifiers, surface tension modifiers and density modifiers, and the like, in order to form “printable inks” that may be delivered in droplets generated by inkjet printhead nozzles. “Printable” in reference to a reagent ink means repeatable droplets are able to be ejected from the nozzle, with uniform velocities and volumes and no satellite droplets. [00102] In some embodiments, the viscosity of a printable reagent composition is less than 8 cP (8 mPa.s^-1) when measured at 20 °C. In some embodiments, the viscosity of the printable reagent composition ranges from 1 cP to 8cP, more preferably from 2 cP to 8 cP when measured at 20°C. In some embodiments, the viscosity of the printable reagent composition ranges from 1 cP, preferably from 2 cP, to 7 cP, including any viscosity within this range, when measured at 20 °C. In some embodiments, the viscosity of the printable reagent composition ranges from 1 cP, preferably from 2 cP, to 3 cP when measured at 20°C. [00103] In some embodiments, the viscosity of a printable reagent composition is comprised between 2.0 and 2.6 mPa.s-1, preferably between 2.2 and 2.4 mPa.s-1 when measured at 20°C. [00104] Suitable methods for measuring the viscosity of the printable reagent composition are known the skilled person in the art. For example, the dynamic viscosity (η) can be measured at room temperature (19.5 - 20.5 °C) using the rotational viscometer Anton Paar ViscoQC 300 equipped with a 7 mL cuvette, with three measurements for each sample, and calculation of the average of said three measurements. The skilled person knows which spindle to select, according to the target viscosity range. [00105] In some embodiments, a printable reagent composition comprises a viscosity modifying agent. Suitable modifying agents include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, glycerol acetate, ethylene glycol, polyethylene glycol (PEG) of different molecular weights, polyethyleneglycol methyl ether (OMe)PEG, polyethylene dimethyl ether (OMe)₂PEG, poly(vinyl alcohol), carboxymethyl cellulose and hydroxyethyl cellulose. In some embodiments, said viscosity modifier is preferably chosen from the group consisting in DMSO, glycerol, PEG, and their combinations. [00106] In some embodiments, the viscosity modifier is DMSO at a concentration comprised between 20v% and 50v%, preferably between 30v% and 40v%, even more preferably of around 35v%. [00107] In some embodiments, the viscosity modifier is glycerol at a concentration comprised between 10v% and 35v%, preferably between 12v% and 20v%. [00108] In some embodiments, the viscosity modifier is PEG at a concentration comprised between 10v% and 35v%, preferably between 12v% and 20v%., even more preferably around 14v%. [00109] In some embodiments, the viscosity modifier is (OMe)PEG at a concentration comprised between 10v% and 35v%, preferably between 12v% and 20v%., even more preferably around 14v%. [00110] In some embodiments, the viscosity modifier is (OMe)₂PEG at a concentration comprised between 10v% and 35v%, preferably between 12v% and 20v%., even more preferably around 14v%. [00111] It is understood by those of ordinary skill that the viscosity ranges achieved by the above DMSO concentration ranges also may be achieved by equivalent concentration ranges of other viscosity modifiers. Thus, in some embodiments, a cleavage ink comprises a concentration of a viscosity modifier that produces an equivalent viscosity as DMSO at a concentration in the range of from 20 v% to 50 v%. [00112] In some embodiments, the surface tension of the printable reagent composition is comprised between 15 and 50 mN.m-1, preferably between around 35 and 50 mN.m^-1, preferably between around 35 and 40 mN.m^-1 when measured at 20°C. [00113] Suitable methods for measuring the surface tension of the printable reagent composition are known the skilled person in the art. For example, the surface tension can be measured with AquaPi Plus at room temperature (19.5 – 20.5 °C) using both the Du Nouy measurement and the Wilhelmy measurement averaged over 3 minutes, according to the user manual.^Three measurements for each sample and each method are done and the average value is calculated. [00114] In some embodiments, a printable reagent composition comprises a surface tension modifier. In some cases, the surface tension modifier may be a detergent. Suitable detergents include, but are not limited to, Tween 20, Triton X-100, CHAPS, NP-40, octyl thioglucoside, octyl glucoside and dodecyl maltoside. Additional surface tension modifiers (i.e., surfactants) are disclosed in Buret, LabChip, 12: 422-433 (2012). In some embodiments, the surface tension modifier is selected in the group consisting of Tween 20 and Triton X-100. In some embodiments, printable reagent compositions are formulated as emulsions. In some embodiments, printable reagent compositions comprising endonuclease V are formulated as emulsions. [00115] In some embodiments, the surface tension modifier is Tween-20 at a concentration of around 0.05v%. In some embodiments, the surface tension modifier is Triton X-100 at a concentration of around 0.04v%. [00116] In some embodiments, a printable reagent composition further comprises a buffer. Any suitable buffer that can maintain the appropriate pH may be used. Exemplary buffers include cacodylate, HEPES, MES, Tris, imidazole, ADA, ACES, PIPES, MOPS, MOPSO, BES, TES, DIPSO, MOBS, TAPSO, HEPPSO, POPSO, TEA, EPPS, Tricine, and Bicine. [00117] In some embodiments, the buffer comprises about 10 to about 60 mM Tris. [00118] In some embodiments, the pH of the printable reagent composition is comprised between 8.0 and 9.0, preferably between 8.0 and 8.5, even more preferably around 8.2. [00119] In some embodiments the concentration of MgCl₂ in the printable reagent composition is comprised between 1.5 and 100 mM preferably around 25 mM. [00120] In some embodiments the concentration of NaCl is comprised between 3 and 50 mM, preferably around 6 mM, or the concentration of (NH₄)₂SO₄ is comprised between 3 and 15 mM. [00121] In some embodiments the printable reagent composition can further comprise Bovine Serum Albumin (BSA). Typically, BSA can be present in a concentration ranging from 0.01 to 10 to µg/mL, preferably 0.05 to 5 µg/mL, even more preferably from 0.1 to 1 µg/mL as stabilizing agent. Delivery of Inks using an Inkjet [00122] Inkjet assisted enzymatic synthesis and cleavage of polynucleotides may be implemented in a variety of embodiments in which a set of printable ink reagent compositions is delivered by inkjet printhead nozzles. In some embodiments, the surface of a reaction site comprises a layer of initiator oligonucleotides and is surrounded by a hydrophobic surface of substrate, which allows the reaction site to be enveloped by a volume of aqueous liquid on the surface without spreading or coalescing with liquid from another reaction site. The printable reagent composition ink, as described above, is dispensed as droplets at a reaction site via an inkjet printhead nozzle. The printable reagent composition ink comprises predetermined concentrations of endonuclease V, and may, in addition, include salts and buffer components for endonuclease V activity and viscosity modifiers and surface tension modifiers as needed to meet the rheological requirements for droplet formation. Droplets may also include humectants to minimize evaporation loss. [00123] As noted above, embodiments of the method may include one or more washing steps, wherein a wash solution is flowed or sprayed on a substrate comprising an array of reaction sites. Wash solution may comprise a variety of solvents including, but not limited to, water, acetonitrile, methanol, PBS or other buffered salt solutions, or the like. In some embodiments, a wash solution may include one or more proteases, e.g., proteinase K, for the purpose of removing any polymerases that may adhere to the reaction site. In some embodiments, the method may further include a step of treating reaction sites with one or more proteases to remove or deactivate polymerases that accumulate at the reaction sites. [00124] In some embodiments, substrates comprise reaction sites continuously enveloped by, or occupied by, a droplet. In other embodiments, reaction sites are dried between cycles of steps so that, strictly speaking, the substrate is not always, or not continuously, a droplet microarray throughout a synthesis. [00125] In some embodiments, including those described above, the plurality of polynucleotides enzymatically synthesized (that is, the number of reaction sites) on a substrate with inkjet delivery of reagents is in the range of from 100 to 10 billion, or in the range of from 100 to 10 million, or in the range of from 100 to 100 thousand, or in the range of from 100 to 500 thousand, or in the range of from 1000 to 1 million. In some embodiments, such pluralities are synthesized on a substrate having a surface area in the range of from 1 cm² to 1 m², 1 cm² to 500 cm², or from 1 cm² to 256 cm², 1 cm² to 30 cm², or having a surface area in the range of from 1 cm² to 15 cm², or having a surface area in the range of from 1 cm² to 7 cm², or having a surface area in the range of from 7 cm² to 20 cm². In some embodiments, substrates may be prepared and undergo surface treatment after which it is cut, or diced, into smaller pieces for use. In some embodiments, the lengths of the polynucleotides synthesized in accordance with the invention are in the range of from 10 to 500 nucleotides, or in the range of from 50 to 500 nucleotides, or in the range of from 100 to 400 nucleotides, or in the range of from 100 to 500 nucleotides. In some embodiments, the per cycle coupling efficiency in the synthesis of polynucleotides in these length ranges is at least 98%, or is at least 99%, or is at least 99.5%, or is at least 99.8%, or is at least 99.9%. In some embodiments, the coupling cycle time in the synthesis of polynucleotides in these length ranges is less than 15 min per cycle, or less than 10 min per cycle, or less than 7 min per cycle, or less than 5 min per cycle. [00126] In some embodiments, inkjet delivery of droplets may be directed to features on a substrate which have a dimension directly related to its size or area, such as a width of a square reaction site or a diameter of a round reaction site. Thus, in some embodiments, reaction sites have a width or diameter in the range from about 10 μm to about 1.0 cm. In some embodiments droplets can be deposited to reaction sites whose widths, or diameters, are in the range of from about 1.0 μm to about 1.0 mm, usually about 5.0 μm to 500 μm, more usually about 10 μm to 200 μm, and still more usually from about 20 µm to about 100 µm. Apparatus for Inkjet Synthesis and Cleavage [00127] Delivering fluids by inkjets is a mature technology that has been available for several decades so that extensive literature is available describing it and providing guidance for adapting it to novel applications, such as for inkjet-assisted synthesis of polynucleotides, as described herein. Exemplary references providing guidance for constructing inkjet delivery systems: Lausted et al, Genome Biology, 5: R58 (2004); Le, Recent Progress in Ink Jet Technologies II, chapter 1, pgs. 1-14 (1999); Derby (2010, cited above); Zapka, editor, “Handbook of Industrial Inkjet Printing,” (Wiley-VCH, Weinheim, Germany); U.S. patents 5474796; 10384189; 10669304; 6306; 6323043; 5847105; and the like. As noted by Le (1999) inkjet printhead nozzles may be classified as “continuous” and “drop-on-demand” (DOD). In some embodiments, DOD inkjet printhead nozzles are employed with apparatus of the invention, and in particular, of the various DOD inkjets, piezoelectric inkjet printhead nozzles are of interest. For example, droplet formation in DOD inkjets is described in Dong et al, Physics of Fluids, 18: 072102 (2006). Such varieties of inkjet printhead nozzles are available in banks or assemblies of large numbers of inkjets (e.g. from 10’s to 100’s) that may be individually programmed for actuation and delivery of droplets. Such inkjets and inkjet assemblies (referred to herein as “inkjet heads”) are commercially available from many manufacturers including Epson, Xaar, Fujifilm, and the like. As used herein, “inkjet printhead nozzle” means a device capable of generating and ejecting droplets of a fluid. In some embodiments, an inkjet printhead nozzle is a device capable of generating and ejecting droplets of a fluid at a predetermined rate and of a predetermined uniform size. [00128] In some embodiments, components of an inkjet apparatus may be arranged according to whether they may be moved relative to one another or whether they are fixed. Computer and software provide overall control of the system components, either directly or indirectly via controllers. For example, software may provide for single pass reagent deposition in which print head is stationary and synthesis support holder moves to deliver reagents to reaction sites. Alternatively, different software may provide for one or more moving print heads and/or moving synthesis support holder via a variety of components, such as, a print controller, print head driver and motion controller. Typically, computer and software control capping station, flush station, wiper, inspection system and washing and drying functions are included. A capping station keeps the print head moist and stops drying of ink. A flush station primes and flushes the print head, which helps remove trapped air and debris as well as dried ink. A wiper is used to remove excess ink and prevent cross-contamination. It may be part of the flush station. An inspection system records the presence, absence or size of spots of deposited reagents or incorrectly placed spots of reagents. The inspection system may comprise a camera that takes images of the synthesis support, and image analysis software extracts and processes information from the images. Such information may be used in real- time to optimize synthesis or to implement corrective measures. Washing and drying functions are carried out by a fluid delivery system separate from that used for droplet delivery. Washing may include deprotection steps, wherein a deprotection reagent is flowed across a synthesis substrate, optionally followed by a drying step. Drying may be accomplished by blowing air or an inert gas, such as argon, over the synthesis support, or by using a volatile solvent, such as methanol, in the washing step. [00129] In some embodiments, cameras or microscopes may be used to capture images of the spots (i.e., reaction sites) and identify missing spots, determine spot size and spot placement. Lighting for image capture may be from above, from the side, from below or integrated into a substrate holder, whichever gives the best contrast in the absence or presence of dye in the inks. Where a dye is used, it is selected so that it does not interfere with the enzymatic reaction, does not react with the protecting group of the nucleotide, and is compatible with the enzyme and deprotection buffers. In some embodiments, each composition comprising a monomer has a different distinguishable dye, covering a different part of the visible spectrum. In some embodiments, imaging of an array of reaction site is carried out during incubation (30 s - 10 min) of the elongation reactions and using high enough magnification to see individual spots but not so high that an inordinate length of time would be needed to scan the array. The number of images taken in an imaging step may be 20 to 100 for a standard microscope slide. Images may be captured seamlessly in a video stream by scanning the substrate or captured in a move-stop process. The images captured may be stitched using algorithms and aided by the presence of fiducial markings on the slide. Fiducial markings also help determine whether the slide has moved in the slide holder and help determine spot positions. In some embodiments, real time image analysis allowing the identification of missing spots or poor spot placement could be accompanied by the automatic generation of a new image and an additional print or prints. [00130] In some embodiments, a plurality of DOD inkjets is housed in a print head which is capable of x-y and z movement relative to droplet microarray. In some embodiments both the print head and the droplet microarray are capable of x-y movement. In some embodiments, the print head is held in a fixed position and the droplet array undergoes x-y movement. The print head may further comprise containers containing printable reagent compositions or inks, as well as viscosity and surface tension modifiers, humectants, and the like, as needed to meet the requirements for desired droplet formation and/or to reduce evaporation loss. The print head may also include temperature regulation to maintain the inks at a temperature optimized for delivery and activity. In some embodiments, some reagents are flowed or delivered to the droplet microarray in bulk such as the deprotection solution and wash solutions. The droplet microarray, which is formed on the substrate, sits or is mounted in a flow chamber, which comprises an inlet and outlet. A flow chamber defines the flow path of reagents (not delivered by the print head) over the droplet microarray. Such reagents may flow continuously over the droplet microarray, or reagents may be delivered to the flow chamber where they remain for a predetermined incubation time, and then are removed or recycled. Such reagents may be moved by conventional pumps or by pressure heads over reagent reservoirs. The flow chamber may also include temperature control elements and humidity control elements to maintain or optimize coupling reaction activity. After exiting, reagents are discarded into a waste container or recycled. Timing of inkjet discharges, positioning of print head, actuation of valves is controlled by fluidics/inkjet controller, which may include imaging software that performs analysis of array images obtained by a camera and that causes alterations of reagent deposition, for example, when coalescing reaction sites are detected. In some embodiments, the print head may be driven by electronics available from Meteor (Meteor Inkjet Ltd, Cambridge, UK). For example, a Print Controller Card (PCC) can be used that synchronizes to the encoder signal from a Thorlabs motion controller. A Head Driver Card (HDC) provides power and a waveform to the printhead. The drive electronics are controlled by Meteor’s digital printing front end, which includes MetDrop and MetWave software for optimization of spotting parameters, with printing initiated by the Thorlabs Kinesis software. Overall instrument control can be performed by instrument software, such as LabView. [00131] Typically, the distance between the inkjet nozzles and the substrate surface may be in the range of from about 10 μm to 10 mm, or in the range of from about 100 μm to 2 mm, or in the range of from about 200 μm to 1 mm, or in the range of from 500 µm to 3 mm. Droplet velocities may be in the range 1–10 meters/sec. Print head movement may be in the range of from 1-30 cm/sec, or 5-30 cm/sec, or 20-30 cm/sec. As described more fully below, print heads may have different droplet delivery modes, for example, single-pass mode, multiple pass mode, and move-stop mode. [00132] In some embodiments, inkjet-based synthesizers include droplet detection components to monitor and record any anomalies in droplet formation and delivery by the inkjet nozzles. In some embodiments, such droplet monitoring may comprise a laser diode mounted orthogonally to the direction of print-head motion such that the droplet stream of each bank of nozzles intersects the beam, causing the light to scatter if a droplet is present. Before each round of printing, nozzles may be fired in series through the beam and the forward scattering of each droplet is detected by a photodiode. Nozzles failing to fire may be taken off- line during synthesis. The inkjet apparatus may also be equipped with commercially available droplet monitors, such as, a Meteor dropwatcher, available from Meteor Inkjet Ltd, (Cambridge, UK) as well as a camera to image the solid support and array of reaction sites. The latter permits the array of reaction sites to be monitored to detect accuracy in droplet deposition, size and geometry of reaction sites, coalescence of reaction sites, and the like. In some embodiments, software may be provided to provide a full image of an array on a slide or solid support by patching together tiles comprising smaller images, e.g. S. Preibisch, S. Saalfeld, P. Tomancak, Bioinformatics, 2009, 25(11), 1463-1465. [00133] In some embodiments, it may be desirable to prevent evaporation of the synthesis reagents and reaction mixtures following deposition. Evaporation may be prevented in a number of different ways. In some embodiments, synthesis cycles may be carried out in a high humidity environment, such as a relative humidity in the range of from 75-85%. Alternatively, or in addition to, one may employ reagents with an evaporation retarding agent or humectant, e.g. glycerol, glycerol acetate, polyethylene glycol, carboxymethyl cellulose, hydroxyethyl cellulose, and the like. [00134] In some embodiments, recirculating ink print heads are employed because problems of drying and/or clogging of nozzles by enzymes is reduced. Recirculating ink print heads are commercially available, for example, from Fujifilm and are described in U.S. patents 8820899; 8534807; 8752946; 9144993; 9511598; 9457579, which are incorporated herein by reference. Synthesis Substrates [00135] In some embodiments, substrates for synthesis comprise surfaces that have been patterned with hydrophobic and hydrophilic regions wherein discrete hydrophilic reaction sites are formed. These allow the formation of droplets on hydrophilic reaction sites, for example, after flowing aqueous reagents or reactants of the entire surface. That is, in some embodiments, substrates for synthesis comprise so-called “droplet microarrays,” e.g., as disclosed in the following exemplary references, which are incorporated by reference: Brennan, U.S. patent 5,474,796; Chrisey et al, Nucleic Acids Research, 24(15): 3040-3047 (1996); Fixe et al, Materials Research Society Symposium Proceedings. Volume 723. Molecularly Imprinted Materials - Sensors and Other Devices. Symposia (San Francisco, California on April 2-5, 2002); Goldfarb, U.S. patent publication 2008/0166667; Gopinath et al, ACS Nano, 8(12): 12030-12040 (2014); Hong et al, Microfluid. Nanofluid., 10: 991-997 (2011); Kumar et al, Nucleic Acids Research, 28(14): e71 (2000); Peck et al, U.S. patent 10384189; Indermuhle et al, U.S. patent 10669304; Wu et al, Thin Solid Films, 515: 4203-4208 (2007); Zhang et al, J. Phys. Chem., 111: 14521-14529 (2007): and like references. As used herein, the term “droplet microarray” refers to a planar substrate whose surface has been treated to create a plurality of discrete hydrophilic regions, which may serve as reaction sites either directly or with further treatment, e.g. attaching initiators. In some embodiments, each of the plurality of discrete hydrophilic regions are surrounded by hydrophobic regions. The discrete hydrophilic regions may have a variety of shapes but are usually circular or rectangular or square for manufacturing convenience. In some embodiments, reaction sites have areas and capacities to hold an aqueous reaction mixture as described above. Although synthesis substrates of some embodiments may comprise droplet microarrays, in a synthesis process such arrays may undergo a drying step which removes liquid from reaction sites. That is, in some embodiments, a synthesis substrate comprising a droplet microarray may be devoid of droplets from time to time, for example, after an elongation cycle ending in a drying step. The hydrophilic-hydrophobic configurations permit the formation of droplets on the surface of a droplet microarray either after inkjet delivery of a synthesis reagent to the hydrophilic regions or by flowing a “bulk” aqueous solution, such as a synthesis reagent or wash solution, over the substrate. As disclosed in the above references, the droplets retained by the hydrophilic regions may serve as reaction chambers or vessels. The planar substrate has a surface with hydrophobic region and discrete hydrophilic regions, which may serve as reaction sites. When the planar substrate is flooded with an aqueous solution both hydrophobic regions and hydrophilic regions are immersed. When the aqueous solution drains off, some of the aqueous solution is retained by hydrophilic regions to form droplets of the droplet microarray. Individual droplets may be referred to as a “microarray droplet” to distinguish them from droplets formed by an inkjet printhead nozzle prior to its delivery to a reaction site. [00136] Preparation of substrates with discrete reaction sites can be accomplished by known methods. For example, such methods can involve the creation of hydrophilic reaction sites by first applying a protectant, or resist, over selected areas over the surface of a substrate, such as a silicon oxide, or like material. The unprotected areas are then coated with a hydrophobic agent to yield an unreactive surface. For example, a hydrophobic coating can be created by chemical vapor deposition of (tridecafluorotetrahydrooctyl)-triethoxysilane onto the exposed oxide surrounding the protected circles. Finally, the protectant, or resist, is removed exposing the well regions of the array for further modification and nucleoside synthesis using the high surface tension solvents described herein and procedures known in the art such as those described by Maskos & Southern, Nucl. Acids Res. 20:1679-1684 (1992). Alternatively, the entire surface of a glass plate substrate can be coated with hydrophobic material, such as 3- (1,1-dihydroperfluoroctyloxy)propyltriethoxysilane, which is ablated at desired loci to expose the underlying silicon dioxide glass. The substrate is then coated with glycidyloxypropyl trimethoxysilane, which reacts only with the glass, and which is subsequently “treated” with hexaethylene glycol and sulfuric acid to form an hydroxyl group-bearing linker upon which chemical species can be synthesized (Brennan, U.S. Pat. No. 5,474,796). Arrays produced in such a manner can localize small volumes of solvent within the reaction site by virtue of surface tension effects (Lopez et al., Science 260:647-649 (1993)). [00137] In some embodiments, reaction sites may be formed on a substrate following the photolithographic methods of Brennan, U.S. patent 5474796; Peck et al, U.S. patent 10384189; Indermuhle et al, U.S. patent 10669304; Fixe et al (cited above); or like references cited above. In accordance with these methods, a set of hydrophilic molecules comprising an aminosilane is attached to the surface of a substrate to form reaction sites. Such hydrophilic molecules may comprise N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11- acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3- aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane (GOPS), or 3-iodo- propyltrimethoxysilane. A set of hydrophobic molecules comprising a fluorosilane is attached to the surface of the substrate in regions outside of the reaction sites. Such hydrophobic molecules may comprise perfluorooctyltrichlorosilane octylchlorosilane, octadecyltrichlorosilane, (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane, or tridecafluoro-1,1,2,2-tetrahydrooctyl)trimethoxysilane. After such attachment, a substrate is prepared for polynucleotide synthesis by coupling initiators to the aminosilanes at the reaction sites. Such coupling may be accomplished using any number of available homo- or heterobifunctional linkers to form covalent bonds between amino groups on the substrate and 5’-thiol groups or 5’-amino groups on the initiators. Such linkers are, for example, available from Sigma-Aldrich (St. Louis, MO) and are described in treatises such as, Hermanson, Bioconjugate Techniques, 3^rd Edition (Academic Press, 2013). Synthesis of oligonucleotides having 5’-thiol or 5’-amino groups is well-know and is described in Kupihar et al, Nucleosides Nucleotides & Nucleic Acids, 22(5-8): 1297-1299 (2003); Fung et al, U.S. patent 4757141; and like references. [00138] In some embodiments, an array of reaction sites may be formed using click chemistry by depositing under coupling conditions droplets of 5’-DBCO (dibenzocyclooctyl) labeled initiators (e.g. Glen Research) on a planar substrate comprising an azide layer (e.g. PolyAn 2D azide glass slide). In some embodiments, such reactions may be carried out as a copper-free click reaction which is less damaging to the DNA, e.g. Dommerholt et al, Top. Curr. Chem. (Z) 374: 16 (2016). [00139] A wide variety of substrates may be employed for creating arrays of reaction sites for enzymatic synthesis of polynucleotides. Substrates may be a rigid material including, without limitation, glass; fused silica; silicon such as silicon dioxide or silicon nitride; metals such as gold or platinum; plastics such as polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and any combination thereof. A rigid surface can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass. Substrates may also comprise flexible materials, which is capable of being bent, folded or similarly manipulated without breakage. Exemplary flexible materials include, without limitation, nylon (unmodified nylon, modified nylon, clear nylon), nitrocellulose, polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene, acetal, acrylic, acrylonitrile, butadiene styrene (ABS), polyester films such as polyethylene terephthalate, polymethyl methacrylate or other acrylics, polyvinyl chloride or other vinyl resin, transparent PVC foil, transparent foil for printers, Poly(methyl methacrylate) (PMMA), methacrylate copolymers, styrenic polymers, high refractive index polymers, fluorine-containing polymers, polyethersulfone, polyimides containing an alicyclic structure, rubber, fabric, metal foils, and any combination thereof. [00140] In some embodiments, patterned surfaces of superhydrophobic and superhydrophilic regions may be formed on a substrate. Guidance for forming droplet microarrays with such patterned surfaces are described in the following references, which are incorporated by reference: Feng et al, Adv. Mater. Interfaces, 1400269 (2014); Zhan et al, Trends Anal. Chem., 108: 183-194 (2018); Neto et al, Adv. Functional Mater., 201400503 (2014). [00141] Achieving accurate alignment of droplet delivery to reaction sites of a prefabricated droplet microarray is an important aspect of inkjet-assisted synthesis of polynucleotides. In some embodiments, such alignment tasks may be minimized or avoided by creating immediately prior to synthesis an array of reaction sites by depositing droplets of synthesis reagents onto a layer of initiator oligonucleotides on a substrate in order to define the locations of reaction sites. Following this initial deposit of droplets, the initiator layer outside of the droplet-defined sites are treated to render them inert to subsequent extension or to render them inert to extension as well as hydrophobic. After such an initial surface treatment to create reaction sites, further or subsequent inkjet delivery of droplets to the same reaction sites will be accurate because the same inkjet printhead nozzles that were used to define the locations of the reaction sites will be used to deliver subsequent droplets during synthesis of the polynucleotides. In some embodiments, the synthesis reagents delivered to the initiator layer comprise a mixture of a template-free polymerase and a 3’-O-protected-dNTP. These reagents extend the initiators to define reaction sites or regions on the oligonucleotide layer which is populated by extended fragments having 3’-O-protected ends. The areas outside of these regions are then treated to render them inert to extensions. In some embodiments, after the initial coupling step defining reaction sites, the entire substrate is exposed to a template-free polymerase and a terminator, such as a dideoxynucleoside triphosphate (ddNTP), or like reagent. In some embodiments, such ddNTP could be, for example, a ddNTP conjugated to a hydrophobic moiety, thereby rendering the coating outside of the reaction sites hydrophobic. Such a hydrophobic moiety may be, for example, a dye or quencher molecule, such as, a Black Hole Quencher® molecule. A variety of terminators may be employed for this purpose. In particular, terminators include nucleoside triphosphates that lack a 3’-hydroxyl substituent and include 2',3'-dideoxyribose, 2',3'-didehydroribose, and 2',3’-dideoxy-3’-haloribose, e.g. 3’- deoxy-3’-fluoro-ribose or 2’,3'-dideoxy-3’-fluororibose nucleosides. Alternatively, a ribofuranose analog can be used in terminators, such as 2',3'-dideoxy-β-D-ribofuranosyl, β-D- arabinofuranosyl, 3'-deoxy-β-D-arabinofuranosyl, or the like. Further terminators are disclosed in the following references: Chidgeavadze et al., Nucleic Acids Res., 12: 1671-1686 (1984); Chidgeavadze et al., FEBS Lett., 183: 275-278 (1985); Izuta et al, Nucleosides & Nucleotides, 15: 683-692 (1996); and Krayevsky et al, Nucleosides & Nucleotides, 7: 613-617 (1988). Nucleotide terminators also include reversible nucleotide terminators, e.g. Metzker et al. Nucleic Acids Res., 22(20):4259 (1994). Methods of Inkjet-assisted Enzymatic Cleavage of polynucleotides [00142] After synthesis is completed, polynucleotides with the desired nucleotide sequence may be released from initiators and the synthesis supports by enzymatic cleavage using an endonuclease V. [00143] Accordingly, the present invention relates to method for enzymatically cleaving a plurality of polynucleotides each comprising a deoxyinosine on a substrate, wherein the substrate comprises a plurality of reaction sites and each polynucleotide of the plurality is assigned to a reaction site; wherein the method comprises dispensing through one or more inkjet printhead nozzles at least one droplet of a printable reagent composition comprising an endonuclease V to each reaction site of the plurality or subset thereof. [00144] In one embodiment, the method for enzymatically cleaving a plurality of polynucleotides each comprising a deoxyinosine on a substrate allows cleaving only a subset of polynucleotides, present in a subset of reactions sites of the substrate. [00145] In one embodiment, the method for enzymatically the cleaving of a plurality of polynucleotides each comprising a deoxyinosine on a substrate comprises dispensing a printable reagent composition comprising Endonuclease V to each reaction site or to a subset of reaction sites, wherein the amount of Endonuclease V dispensed to each reaction site is not identical across the different reaction sites. This can be done either by varying the amount of printable reagent composition comprising endonuclease V and/or by varying the concentration of Endonuclease V in the printable reagent composition. Advantageously, these embodiments allow the recovery of different amounts of each polynucleotide, which can be desirable when the downstream use of said polynucleotides requires non-stochiometric amounts of each polynucleotide. [00146] In other terms, the methods of the invention are customizable (i.e. they can allow the recovery of different amounts of each polynucleotide, depending on the desired subsequent use thereof). [00147] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. EXAMPLES [00148] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the disclosed subject matter. The examples below are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. It should be understood, however, that the intention is not to limit the invention to the particular embodiments described. The intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. [00149] Unless specified to the contrary, methods for enzymatically synthesizing a plurality of polynucleotides are carried out as described in Verardo et al. Sci. Adv. 9, eadi0263 (2023), except that the initiator/primer did not contain a UV-cleavable group for post-synthesis release by photocleavage. Example 1: Endo V printing /cleaving test [00150] In previous approaches of inkjet assisted synthesis of polynucleotides, recovery of the synthesized polynucleotides was carried out by bulk cleavage as follows: - addition of Cleaving solution (1.4 mL buffer (170 mM NaCl, 50 mM MgCl2, 10 mM Tris) in gasket, - denaturation of EndoV at 98°C, - evaporation of the sample via speedvac, - resuspension into 10 µL biological water. [00151] The final salt concentration of the recovered polynucleotide solution was 23.8 M NaCl, 7 M MgCl₂, 1.4 M Tris, 108 µM EndoV, which is not compatible with any downstream PCR steps. [00152] Post-synthesis purification steps were therefore required before carrying out nucleic acid amplification using said polynucleotides. [00153] Here, the inventors have shown that is possible to carry out the cleavage step via inkjet printing of a formulation comprising Endo V, according to a specific pattern. [00154] The Endo V printing was carried out as follows: - cleaving ink (total per slide of about 6.5 µL, various formulations as detailed below) was printed via inkjet on the slide at room temperature and 70% relative humidity (RH), - the slide with the cleaving ink was incubated for 1 h at 45°C and at least 85% RH, - denaturation of EndoV at 98°C, - evaporation of the sample via speedvac, - resuspension into 10 µL biological water. [00155] The final salt concentration of the recovered polynucleotide solution depended on each formulation but was much lower than in the previous approach, due to the fact that the volume of cleaving solution was significantly reduced. [00156] In addition, DNA recovery could be carried out in a pattern-specific manner, with cleaving yields above 80%. [00157] Figure 1 shows a microscope image and schematics of an iDNA printed slide that was consecutively EndoV-cleaved via printing with a specific pattern. The experiment displays no cross-contamination between the sites that were meant to be cleaved and were successfully cleaved and the sites that were not meant to be cleaved and were successfully preserved. [00158] Figure 2 is a Typhoon image related to the experiment of Figure 1, as well as the corresponding calculated cleaving yields. Example 2: Printable formulations for Inkjet Enzymatic DNA Cleavage [00159] For successful inkjet enzymatic DNA cleavage, the buffer containing the enzyme (referred to as an “ink”) is preferably active (i.e., result in efficient cleavage); stable (i.e., does not form a precipitate that may clog the printhead); and printable (i.e., has appropriate viscosity (η) and surface tension (γ)). [00160] Experiments were first carried out with a standard cleavage buffer consisting of 10 mM Tris, 170 mM NaCl and 50 mM MgCl2, pH 8, with or without DMSO and/glycerol as viscosity modifiers, and using different amounts of Endo V enzyme. These experiments were carried out in bulk solution. [00161] The Endo V enzyme used throughout the Examples is a synthetic variant of Endo V having the sequence set forth in SEQ ID NO:3 (which corresponds to the synthetic polypeptide having SEQ ID NO:2 with an N-terminal His-tag), but similar results are obtained with other variants of Endo V. [00162] Table 1 below represents surface cleaving yields for Endo V cleavage inks (“EV inks”) compared to standard condition (no viscosity modifier). Table 1: S † Temperature lide cleaving yields (%, ± sd, n = 2)** [Endo V]* /°C 10% gly. + 15% Standard 35% DMSO DMSO 0.5x Not calculated 68.6 ± 0.3 70.4 ± 1.6 20 1x 71.2 ± 6.3 67.2 ± 0.5 70.2 ± 0.4 5x Not calculated 64.8 ± 3.3 69.4 ± 0.1 0.5x Not calculated 62.6 ± 1.7 60.7 ± 3.6 37*** 1x 54.7 ± 2.8 58.9 ± 0.1 60.3 ± 2.0 5x Not calculated 54.0 ± 1.7 56.1 ± 0.7 0.5x Not calculated 73.7 ± 1.8 73.9 ± 1.9 45 1x 66.2 ± 1.0 73.4 ± 1.6 72.5 ± 1.9 5x Not calculated 70.5 ± 0.4 67.9 ± 0.6 Incubation time of 1 h for 37 and 45 °C and 20 h for 20°C *) 1x = 0.78µM of EndoV. †) Surface cleaving yield calculated based on Typhoon scans. **) Standard deviation calculated on for n = 2. ***) Test performed on a 16-well gasket instead of an 8-well gasket. [00163] At 45°C, which was selected as an optimal temperature for the cleaving step, DMSO performed as well as a mixture of glycerol/DMSO. [00164] The viscosity of the EV ink was between 2.2 and 2.4 mPas.s^-1. [00165] Then, different surfactants were used as surface tension modifiers, as illustrated in Table 2. Table 2: Surfactant Slide cleaving yields (%, ± error*) Standard condition 71.6 ± 4.1 0.04 v% Triton X-100 71.7 ± 4.0 0.05v% Tween20 74.6 ± 4.0 Cleaving at 20 °C and 0.78 µM EndoV concentration with 35% DMSO for 20 h. *) Error propagation on standard deviations of fluorescence values [00166] Tween-20 was selected as surface tension modifier for further experiments, but Triton X-100 can also be used, with similar results. [00167] The surface tension of the EV ink was between 35 and 40 mN.m^-1. [00168] The inventors have shown that addition of surfactant and/or surface tension modifiers did not adversely affect the cleaving efficacy of Endonuclease V. Example 3: Printable formulations for optimized Inkjet Enzymatic DNA Cleavage [00169] Based on the conditions determined in Example 2 (Tris-based buffer with 35% v/v DMSO,0.05 % v/v Tween-20 and 0.78 µM of Endo V having SEQ ID NO:3), different ink formulations were tested, as described in Table 3 below. Table 3: EV EV EV EV EV EV EV EV EV EV EV EV EV EV EV ink ink ink ink ink ink ink ink ink ink ink ink ink ink ink -1 -3 -4 -5 -6 -7 -8 -9 -10 -11 -12 -13 -14 -15 -16 MgCl2 (mM) 50 3 3 25 50 100 3 25 50 3 3 50 50 25 50 Tris-HCl 10 60 60 60 60 60 60 60 60 30 5 10 10 60 60 (mM) NaCl (mM) 170 15 15 6 3 1.5 - - - 15 15 50 10 - - (NH2)4SO4 (mM) - - - - - - 15 6 3 - - - - 15 15 BSA (µg/mL) - - 1 - - - - - - - - - - - - pH 8.2 8.2 8.2 8.2 8.2 8.2 8.2 8.2 8.2 8.2 8.2 8.1 8.3 8.3 8.3 Cleavage activity tests on surface [00170] The cleaving yield was determined for each formulation ink. Results are shown in Table 4 below. Table 4: Formulation Yield Control - EV-ink1 86 ± 6 EV-ink3 74 ± 4 EV-ink4 74 EV-ink5 80 EV-ink6 83 ± 3 EV-ink7 87 EV-ink8 75 ± 7 EV-ink9 78 ± 2 EV-ink10 80 EV-ink11 76 EV-ink12 72 ± 6 EV-ink13 81 EV-ink14 80 EV-ink15 83 EV-ink16 86 [00171] Figure 3 shows the effect of the concentration of MgCl2, NaCl or (NH4)2SO4, Tris- HCl on the cleavage yield. It can be seen that the cleaving yield increases with the MgCl2 concentration in the range tested of from 3 mM to 100 mM. NaCl and (NH₄)₂SO₄ had similar effects, and reducing their concentration was not deleterious for surface cleavage yield. Reducing the concentration of Tris-HCl was not deleterious for surface cleavage yield. Finally, addition of BSA had no effect on surface cleavage yield. Cleavage activity tests in solution based on qPCR [00172] The different formulation inks were also tested for cleaving activity in solution, based on quantitative PCR. [00173] In this assay, a substrate comprising a deoxyinosine (dI) was subjected to cleavage in 100 µL of each EV formulation ink and compared with the respective EV formulation ink without Endo V. The resulting solutions were then subjected to amplification by qPCR and the average Cq and deltaCq were determined for each ink. [00174] As shown in Figure 4, the most active ink was EVink-15. Downstream amplification tests [00175] The inventors then tested the compatibility of the different cleavage ink formulations with downstream nucleic acid amplification steps, performed on the recovered nucleic acids, without desalting steps (i.e. without post-synthesis purification steps). [00176] PCR amplification of the following sequence GAACTTCAACTTCAACGGCCTTACCGGCACCGGCGTGCTGACCGAGAGCA (SEQ ID NO:4;, also known as E13) was performed and the reaction products were analysed using an Agilent 4150 System (G2992A) and the D1000 screen Tape kit. [00177] The results the most comparable to MQ (milliQ water, used as a reference of low salt conditions known to be favourable for the downstream PCR) were obtained with EVi-5 and EVi-8. In contrast, the PCR product amplified after cleavage with the EVi-16 ink displayed the wrong length. The other inks all performed correctly. [00178] Next-Generation Sequencing (NGS) was then carried out on the amplified PCR product and analysed for average deletion, insertion, substitution and total error rate. The results were satisfactory. [00179] Therefore, the inventors have shown that the method of the invention was compatible with downstream amplification steps, without the need for any desalting step prior to said amplification. [00180] Alternatively, a desalting step can be carried out as follows: Endo V is denatured at 95°C for 5 minutes. The solution comprising the polynucleotides and denatured Endo V is filtered through an 3kDa filter (eg from Amicon®) for 20 minutes at 14,000 rcf. The polynucleotide and denatured enzyme are retained, whereas the salts are discarded. The filter is then reversed and the polynucleotides collected with 50µL MQ for 2 min at 2,000 rcf. This step is done twice. The final solution is concentrated by Speedvac (45 min , 45°C) and resuspended in 10µL of nuclease-free water. The inventors have found that the resulting desalted solution could then be used for subsequent amplification reactions with good results. Stability of the Endo V cleavage inks [00181] Experiments were carried out to find which ink maintains the activity the longest when stored at 20°C. Indeed, since the ink is loaded in the printhead in a mL quantity, and used in an amount of approximately 6.5 µL per slide, it is important to develop cleavage ink formulations that are stable over time when stored at 20°C. [00182] The stability of the inks was first tested in solution, by determining the relative Endo V activity using a FRET test. [00183] Briefly, dual-labelled probes comprising a FRET fluorophore on one end and a FRET quencher on the other end were incubated with the Endo V cleavage inks. Cleavage by Endo V releases the fluorophore from the quencher, resulting in increased fluorescence. [00184] The residual Endo V activity relative to the Endo V activity at time 0 was determined for each ink. [00185] As shown in Figure 5 formulations EVi-5, EVi-6, EVi-15 and EVi-17 were relatively stable over 2 weeks. In particular, EVi-5 maintained excellent Endo V activity after 2 weeks of storage. [00186] The same formulations were also tested for their residual activity after 2 weeks of storage at 20°C by evaluating the surface cleaving yield by Typhoon scan. The results are presented in Table 5 below. Table 5 – Surface cleaving yield in % Evi-1ink EVi-5 EVi-6 EVi-15 EVi-17 Fresh 81 ± 1 79 ± 0 79 ± 1 78 ± 2 79 ± 2 1w 82 79 78 77 76 2w 80 79 80 80 81 [00187] The surface Endo V activity of the EVi-5, EVi-6, EVi-15 and EVi-17 formulation were stable after 1 or 2 weeks of storage at 20°C. Effect of BSA on EVi- formulations [00188] No effect of BSA was observed on cleavage yield, downstream PCR amplification or ink stability, as shown in Figure 6. [00189] The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, accessions, references, databases, and patents cited herein are hereby incorporated by reference for all purposes.

Claims

CLAIMS 1. A method for enzymatically cleaving a plurality of polynucleotides or a subset thereof, each comprising a deoxyinosine on a substrate, wherein the substrate comprises a plurality of reaction sites and each polynucleotide of the plurality is assigned to a reaction site; the method comprising dispensing through one or more inkjet printhead nozzles at least one droplet of a printable reagent composition comprising an endonuclease V to each reaction site of the plurality or to a subset of said reaction sites.

2. A method for enzymatically synthesizing a plurality of polynucleotides each having a predetermined sequence at reaction sites on a substrate, the method comprising: (a) providing the substrate, wherein the substrate comprises initiators at a plurality of reaction sites, wherein each initiator comprises a deoxyinosine penultimate to a 3’-terminal nucleotide having a free 3’-hydroxyl, and wherein each polynucleotide of the plurality is assigned to a reaction site for synthesis; (b) providing a set of printable reagent compositions comprising a template-free polymerase and a 3’-O-protected nucleoside triphosphate, (c) performing a reaction cycle comprising the steps of i) dispensing through one or more inkjet printhead nozzles at least one droplet of one of the printable reagent compositions to each reaction site of the plurality where the 3’-O-protected nucleoside triphosphate is to be added, wherein the initiator or elongated fragments having free 3’-O-hydroxyls are reacted with the 3’-O-protected nucleoside triphosphate under suitable conditions for elongation by the template-free polymerase, wherein the initiator or elongated fragments are elongated by incorporation of the 3’-O-protected nucleoside triphosphate to form 3’-O-protected elongated fragments, and (ii) dispensing through one or more inkjet printhead nozzles at least one droplet of a deprotection solution to deprotect the 3’-O-protected elongated fragments to form elongated fragments having free 3’-hydroxyls; (d) repeating step (c) until the plurality of polynucleotides is synthesized; and (e) dispensing through one or more inkjet printhead nozzles at least one droplet of a printable reagent composition comprising an endonuclease V to each reaction site of the plurality or a subset of said reaction sites.

3. The method of any of the above claims, wherein the printable reagent composition comprising an endonuclease V is filtered through a filter before said dispensing, preferably the filter has a pore size less than or equal to 0.8 µm in diameter.

4. The method of any of the above claims, wherein the printable reagent composition comprising an endonuclease V has a viscosity comprised between 2.2 and 2.4 mPa.s^-1 when measured at 20°C.

5. The method of any one of the above claims, wherein the printable reagent composition comprising an endonuclease V has a surface tension comprised between 35 and 40 mN.m^-1 when measured at 20°C.

6. The method of any one of the above claims, wherein the plurality of polynucleotides obtained by said method can be used in a subsequent nucleic acid amplification step without the need for a desalting step prior to said amplification step.

7. A printable reagent composition comprising - an endonuclease V, preferably an endonuclease V selected from the group consisting of polypeptides having at least 80% identity with wild-type Endo V from E. coli (SEQ ID NO:1) and variants having at least 80% identity with the synthetic variant having the sequence set forth in SEQ ID NO:2, - a buffer, - a viscosity modifier, - a surface tension modifier, - MgCl_2, - a salt selected from NaCl and (NH4)2SO4 - and optionally BSA.

8. A printable reagent composition according to claim 7 wherein the viscosity modifier is selected from dimethyl sulfoxide (DMSO), glycerol and polyethylene glycol (PEG), preferably the viscosity modifier is DMSO at a concentration of around 35v%.

9. The printable reagent composition according to claim 7 or 8, wherein the surface tension modifier is selected from polyoxyethylene (20) sorbitan monolaurate (Tween ® 20) and polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (also known as Octyl phenol ethoxylate or Triton X-100 ®), preferably the surface tension modifier is Tween® 20 (polyoxyethylene (20) sorbitan monolaurate) at a concentration of around 0.05v %.

10. The printable reagent composition according to any of claims 7 to 9, wherein the endonuclease V has a concentration of 0.5 µM to 1 µM, preferably between 0.75 and 0.80 µM, even more preferably around 0.78 µM.

11. The printable reagent composition of any one of claims 7 to 10, wherein the buffer is Tris-HCl, preferably Tris-HCl at a concentration comprised between 10 and 60 mM.

12. The printable reagent composition of any one of claims 7 to 11, wherein said composition has a pH ranging from 8.0 to 9.0, preferably between 8 and 8.5, even more preferably around 8.2.

13. The printable reagent composition of any one of claims 7 to 12, wherein the concentration of MgCl2 is comprised between 1.5 and 100 mM preferably around 25 mM.

14. The printable reagent composition of any one of claims 7 to 13, wherein the concentration of NaCl is comprised between 3 and 50 mM, preferably around 6 mM, or the concentration of (NH4)2SO4 is comprised between 3 and 15 mM. .