Method for enzymatic synthesis of polynucleotides

Abstract

The present invention relates to a method of synthesizing a polynucleotide, wherein the method comprises the steps of (a) performing a cycle comprising the steps of (i) contacting a template- independent polymerase with an initiator nucleic acid and a 3'-O-protected nucleoside triphosphate, so that the initiator nucleic acid is elongated by the template-independent polymerase activity by incorporation of the 3' -O-protected nucleoside triphosphate, to form a 3'-O-protected elongated initiator nucleic acid, (ii) deprotecting the 3'-O-protected elongated initiator nucleic acid to form an elongated nucleic acid having a free 3'- hydroxyl, (b) repeating cycles of step (a) to further elongate the initiator nucleic acid, until the polynucleotide is synthesized, wherein the template-independent polymerase is anchored to a solid support during cycles of step (a). The invention further relates to polymerases, kits, solid supports, devices for use in the invention and related uses.

Description

METHOD FOR ENZYMATIC SYNTHESIS OF POLYNUCLEOTIDESFIELD OF THE INVENTION

[0001] The present invention relates to methods for synthesizing polynucleotides and associated reagents, devices and kits.BACKGROUND

[0002] The interest in polynucleotide synthesis has surged, driven by the growing demand for synthetic polynucleotides in diverse fields. Moreover, chemical methods of polynucleotide synthesis suffer from a number of limitations. These limitations include upper limits on product length, the utilization of moisture-sensitive monomers, and the reliance on environmentally unfriendly solvents, as highlighted by Jensen et al in Biochemistry (57: 1821-1832, 2018).

[0003] Enzymatic approaches for DNA and RNA synthesis have emerged as a promising alternative to chemical synthesis. Current approaches for enzymatic synthesis use template-free polymerases. These enzymes facilitate iterative cycles wherein an initiator polynucleotide undergoes elongation by coupling with a 3'-O-protected nucleotide. The process involves subsequent deprotection of the obtained protected growing strand, continuing until a polynucleotide having a desired sequence is formed. Engineered TdT variants, as described in any one of WO2017 / 216472, W02019 / 135007, W02020 / 099451, WO2021 / 116270, WO2021 / 213903, WO2022 / 063835, WO2023083997, WO2020 / 239737 and WO2023 / 083999 have demonstrated efficient incorporation of reversibly protected 3’-O-protected nucleoside triphosphates.

[0004] The current approaches to enzymatic synthesis are however still limited by several drawbacks. One of them is the fact that the polymerase creates clogging within the tubing of the synthesis apparatus. As a consequence, the tubing must be regularly cleaned or even changed, leading to increased maintenance costs and overall costs for the enzymatic synthesis of DNA or RNA. The cost of the reagents, particularly of the polymerase used in the process, is another drawback. There is therefore a need to further improve the existing DNA / RNA enzymatic synthesis processes.SUMMARY OF THE INVENTION

[0005] In one aspect, the invention relates to a method of synthesizing a polynucleotide, wherein the method comprises the steps of:(a) performing a cycle comprising the steps of:(i) contacting a template-independent polymerase with an initiator nucleic acid and a 3’-O-protected nucleoside triphosphate, so that the initiator nucleic acid is elongated by the template-independent polymerase activity by incorporation of the 3’ -O-protected nucleoside triphosphate, to form a 3 '-O-protected elongated initiator nucleic acid,(ii) deprotecting the 3 '-O-protected elongated initiator nucleic acid to form an elongated nucleic acid having a free 3’ - hydroxyl,(b) repeating cycles of step (a) to further elongate the initiator nucleic acid, until the polynucleotide is synthesized, wherein the template-independent polymerase is anchored to a solid support during cycles of step (a).

[0006] In another aspect, the invention relates to a template-independent polymerase, for use in the method of the invention, wherein said polymerase comprises a moiety with templateindependent polymerase activity and a tag moiety for attaching the polymerase to a substrate initiator nucleic acid.

[0007] In another aspect, the invention relates to a solid support for synthesis of a polynucleotide, wherein the solid support comprises, anchored to the solid support: at least one template-independent polymerase, in particular wherein the polymerase is according to the invention; and optionally at least one initiator nucleic acid, for use as substrate of the template-independent polymerase.

[0008] In another aspect, the invention relates to a kit for performing the method of the invention, comprising: a template-independent polymerase, wherein said polymerase comprises a moiety with template-independent polymerase activity and a tag moiety for attaching the polymerase to an initiator nucleic acid; andan initiator nucleic acid, wherein said initiator nucleic acid comprises a capture moiety for attaching the initiator nucleic acid to the template-independent polymerase; wherein the tag moiety of the polymerase binds to the capture moiety of the initiator nucleic acid.

[0009] In another aspect, the invention relates to a device for performing the method of the invention, wherein said device comprises: a reaction chamber comprising a solid support to which a template-independent polymerase is anchored; and means for dispensing one or more buffer, reagent and / or solvent to the reaction chamber.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Fig. 1 shows a schematic representation of steps of an embodiment of the method of the invention.

[0011] Fig. 2 shows an agarose gel on which polynucleotides synthesized according to the method of the invention are apparent.

[0012] Fig. 3 shows an agarose gel on which polynucleotides synthesized according to the method of the invention are apparent.

[0013] Fig. 4 shows error rate levels of polynucleotides synthesized according to the method of the invention in comparison to polynucleotides synthesized according to control conditions.

[0014] DETAILED DESCRIPTION OF THE INVENTION

[0015] In one aspect, the invention relates to a method of synthesizing a polynucleotide, wherein the method comprises the steps of:(a) performing a cycle comprising the steps of:(i) contacting a template-independent polymerase with an initiator nucleic acid and a 3’-O-protected nucleoside triphosphate, so that the initiator nucleic acid is elongatedby the template-independent polymerase activity by incorporation of the 3’ -O-protected nucleoside triphosphate, to form a 3 '-O-protected elongated initiator nucleic acid,(ii) deprotecting the 3 '-O-protected elongated initiator nucleic acid to form an elongated nucleic acid having a free 3’ - hydroxyl,(b) repeating cycles of step (a) to further elongate the initiator nucleic acid, until the polynucleotide is synthesized.

[0016] According to a preferred aspect of the invention, the template-independent polymerase is anchored to a solid support during cycles of step (a). The inventors have shown that templateindependent polymerases could be anchored and maintain high efficiency for oligonucleotide synthesis. Anchoring the template-independent polymerase for several cycles of oligonucleotide extension provides several advantages. A major advantage is that the synthesis consumes lower amounts of polymerase since it does not require fresh addition of polymerase at each extension cycle. Polymerase is a major source of costs in enzymatic oligonucleotide synthesis and, consequently, the method of the invention offers important cost savings. Anchoring the template-independent polymerase also allows to prevent clogging of the synthesis apparatus, e.g. clogging of the tubes used for supplying reagents. Clogging induces dysfunctions of synthesis and requires special steps for cleaning the tubes and removing the agglomerated polymerase. Clogging may also lead to premature change of the tubing and thus increases the maintenance costs of the synthesis apparatus. Solving clogging issues by anchoring the polymerase thus allows to save costs and time and improves the lifetime of the sequencing apparatus tubing.

[0017] Template-Free Enzymatic Synthesis

[0018] Generally, methods of template-free (or equivalently, “template-independent”) enzymatic DNA synthesis comprise repeated cycles of steps, such as illustrated in Fig. 1, in which a predetermined nucleotide is coupled to an initiator or growing chain in each cycle. The general elements of template-free enzymatic synthesis are described in the following references: Ybert et al, International patent publication WO2015 / 159023; Ybert et al, International patent publication WO2017 / 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: 0.1039 / c6ob01371f (2016); Schmitz et al, Organic Lett., 1(11): 1729-1731 (1999).

[0019] As shown in Figure 1, initiator polynucleotides (100) are provided which have free 3’- hydroxyl groups (103). A template-independent polymerase (104), such as a TdT or variant thereof (e.g. Ybert et al, WO2017 / 216472; Champion et al, W02019 / 135007) is also provided. The template-independent polymerase (104) is anchored to the solid support. To the initiator polynucleotides (100) (or elongated initiator polynucleotides in subsequent cycles) and anchored template-independent polymerase (104) are added a 3’-O-protected-NTP under conditions effective for the enzymatic incorporation of the 3’-O-protected-NTP onto the 3’ end of the initiator polynucleotides (100) (or elongated initiator polynucleotides which are extension products of the initiator polynucleotides). This reaction produces elongated initiator polynucleotides whose 3’-hydroxyls are protected (106). If the elongated initiator polynucleotide does not contain a completed sequence, then the 3’-O-protection groups are removed (108) to expose free 3’-hydroxyls (103) and the elongated initiator polynucleotides are subjected to another cycle of nucleotide addition and deprotection. If the elongated initiator polynucleotide contains a completed sequence, then the 3’-O-protection group may be removed, or deprotected. The desired sequence may be cleaved from the original initiator polynucleotide (110). 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 polynucleotide. An exemplary cleavable nucleotide may be an uracil nucleotide which is cleaved by uracil DNA glycosylase.

[0020] As used herein the terms “nucleic acid”, “polynucleotide”, “oligonucleotide” refers to naturally-occurring or synthetic polymeric forms of nucleotides. The oligonucleotides and nucleic acid molecules of the present invention may be formed from naturally occurring nucleotides, for example forming deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. Alternatively, the naturally occurring oligonucleotides may include structural modifications to alter their properties, such as in peptide nucleic acids (PNA) or in locked nucleic acids (LNA). The solid phase synthesis of oligonucleotides and nucleic acid molecules with naturally occurring or artificial bases is well known in the art. The terms should be understood to include equivalents, analogs of either RNA or DNA made from nucleotide analogs and as applicable to the embodiment being described, single-stranded or doublestranded polynucleotides. Nucleotides useful in the invention include, for example, naturally- occurring nucleotides (for example, ribonucleotides or deoxyribonucleotides), or natural or synthetic modifications of nucleotides, or artificial bases. Any of the nucleic acids disclosed herein may be, in particular embodiments, oligonucleotides.

[0021] In some embodiments, the methods provided herein use nucleic acids that are immobilized on a support or solid support (e.g., support-bound oligonucleotides). As used herein the term “support”, “surface” and “substrate” are used interchangeably and refers to a porous or non-porous solvent insoluble material on which polymers such as nucleic acids are synthesized or immobilized. As used herein “porous” means that the material contains pores having substantially uniform diameters (for example in the nm range). Porous materials include paper, synthetic filters etc. In such porous materials, the reaction may take place within the pores. The support can have any one of a number of shapes, such as pin, strip, plate, disk, rod, bends, cylindrical structure, particle, including bead, nanoparticles and the like. The support can have variable widths. The support can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF) membrane, glass, controlled pore glass, magnetic controlled pore glass, ceramics, metals, and the like etc.; either used by themselves or in conjunction with other materials.

[0022] As used herein, the terms “protected” and “blocked” in reference to specified groups, such as, a 3 ’-hydroxyls of a nucleotide or a nucleoside, are used interchangeably and are intended to mean that a moiety is attached covalently to the specified group to prevent a chemical change to the group during a chemical or enzymatic process. Whenever the specified group is a 3 ’-hydroxyl of a nucleoside triphosphate, or an extended fragment (or “extension intermediate”) in which a 3 ’-protected (or blocked)-nucleoside triphosphate has been incorporated, the prevented chemical change is a further, or subsequent, extension of the extended fragment (or “extension intermediate”) by an enzymatic coupling reaction.

[0023] As used herein, a “self-labelling protein” is a protein which recognizes a small molecule and transfers this small molecule onto itself to form a covalent attachment. An example is the HaloTag which recognizes a haloalkane ligand (Sacca et al., Angew Chem Int Ed Engl. 2010 Dec 3;49(49):9378-83). In some embodiments, the self-labelling protein recognizes a nucleotide, a nucleotide analogue and / or a part thereof. Such self-labelling protein is also called a nucleotide-recognizing self-labelling protein. An example is the SnapTag which recognizes amodified O6-benzylguanine nucleobase (Lindberg, E, Nature communications 9.1 (2018): 3539) or CLIP Tag which recognizes a benzylcytosine. In some embodiments, the nucleotide- recognizing self-labelling protein is a uracyl glycosylase UDGx which forms a covalent conjugate with uracil-DNA.

[0024] As used herein, an “initiator” (or equivalent terms, such as, “initiating fragment,” “initiator nucleic acid,” “initiator oligonucleotide,” or the like) usually refers to a short oligonucleotide sequence with a free 3 ’-hydroxyl at its end, which can be further elongated by a template-free polymerase, such as TdT. In some embodiments, the initiating fragment is a DNA initiating fragment. In an alternative embodiment, the initiating fragment is an RNA initiating fragment. In some embodiments, an initiating fragment possesses between 3 and 100 nucleotides, in particular between 3 and 20 nucleotides. In some embodiments, the initiating fragment is single-stranded. In alternative embodiments, the initiating fragment may be doublestranded. 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.

[0025] In some embodiments, an initiator may comprise a non-nucleic acid compound having a free hydroxyl to which a TdT may couple a 3’-O-protected dNTP, e.g. Baiga, U.S. patent publications US2019 / 0078065 and US2019 / 0078126.

[0026] 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 step after each reaction, or extension, step, as well as after each 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 sec, to several minutes, e.g. 30 min.

[0027] When the sequence of polynucleotides on a synthesis support includes reverse complementary subsequences, secondary intra-molecular or cross-molecular structures may be created by the formation of hydrogen bonds between the reverse complementary regions. In some embodiments, base protecting moieties for exocyclic amines are selected so that hydrogens of the protected nitrogen cannot participate in hydrogen bonding, thereby preventingthe formation of such secondary structures. That is, base protecting moieties may be employed to prevent the formation of hydrogen bonds, such as are formed in normal base pairing, for example, between nucleosides A and T and between G and C. At the end of a synthesis, the base protecting moieties may be removed and the polynucleotide product may be cleaved from the solid support, for example, by cleaving it from its initiator.

[0028] In addition to providing 3’-O-blocked dNTP monomers with base protection groups, elongation reactions may be performed at higher temperatures using thermal stable templateindependent polymerases. For example, a thermal stable template-independent polymerase having activity above 40°C may be employed; or, in some embodiments, a thermal stable template-independent polymerase having activity in the range of from 40-85°C may be employed; or, in some embodiments, a thermal stable template-independent polymerase having activity in the range of from 40-65°C may be employed.

[0029] In some embodiments, elongation conditions may include adding solvents to an elongation reaction mixture that inhibit hydrogen bonding or base stacking. Such solvents include water miscible solvents with low dielectric constants, such as dimethyl sulfoxide (DMSO), methanol, and the like. Likewise, in some embodiments, elongation conditions may include the provision of chaotropic agents that include, but are not limited to, n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2- propanol, sodium dodecyl sulfate, thiourea, urea, and the like. In some embodiments, elongation conditions include the presence of a secondary-structure-suppressing amount of DMSO. In some embodiments, elongation conditions may include the provision of DNA binding proteins that inhibit the formation of secondary structures, wherein such proteins include, but are not limited to, single-stranded binding proteins, helicases, DNA glycolases, and the like.

[0030] 3’ -O-blocked dNTPs with or without base protection may be purchased from commercial vendors or synthesized using published techniques, e.g. U.S. patent 7057026; Guo et al, Proc. Natl. Acad. Sci., 105(27): 9145-9150 (2008); Benner, U.S. patents 7544794 and 8212020; International patent publications W02004 / 005667, WO91 / 06678; Canard et al, Gene (cited herein); Metzker et al, Nucleic Acids Research, 22: 4259-4267 (1994); Meng et al, J. Org. Chem., 14: 3248-3252 (3006); U.S. patent publication 2005 / 037991.

[0031] When base-protected dNTPs are employed, the method may further include a step of removing base protecting moieties, which in the case of acyl or amidine protection groups may (for example) include treating with concentrated ammonia.

[0032] The above method may also include one or more capping steps in addition to washing steps after the reacting, or extending, step. A first capping step may cap, or render inert to further extensions, unreacted 3 ’-OH groups on partially synthesized polynucleotides. Such capping step is usually implemented after a coupling steps, and whenever a capping compound is used, it is selected to be unreactive with protection groups of the monomer just coupled to the growing strands. In some embodiments, such capping steps may be implemented by coupling (for example, by a second enzymatic coupling step) a capping compound that renders the partially synthesized polynucleotide incapable of further couplings, e.g. with TdT. Such capping compounds may be a dideoxynucleoside triphosphate. In other embodiments, non-extended strands with free 3 ’-hydroxyls may be degraded by treating them with a 3 ’-exonuclease activity, e.g. Exo I. For example, see Hyman, U.S. patent 5436143. Likewise, in some embodiments, strands that fail to be deprotected may be treated to either remove the strand or render it inert to further extensions. A second capping step may be implemented after a deprotection step, to render the affected strands inert from any subsequent coupling or deprotection any 3’-0 protection, or blocking groups. Capping compounds of such second capping step are selected so that they do not react with free 3 ’-hydroxyls that may be present. In some embodiments, such second capping compound may be a conjugate of an aldehyde group and a hydrophobic group. The latter group permits separation based on hydrophobicity, e.g. Andrus, U.S. patent 5047524.

[0033] In some embodiments, reaction conditions for an elongation step (also sometimes referred to as an extension step or a coupling step) may comprise the following, in addition to the anchored TdT: 125-600 pM 3’-O-blocked dNTP (e.g. 3’-O-NH2-blocked dNTP); about 10 to about 500 mM potassium cacodylate buffer (pH between 6.5 and 7.5) and from about 0.01 to about 10 mM of a divalent cation (e.g. CoC12 or MnC12), where the elongation reaction may be carried out in a 50 p L reaction volume, at a temperature within the range of from room temperature to about 50°C, for a duration of, e.g., 2-5 minutes. In embodiments, in which the 3’-O-blocked NTPs are 3’-O-NH2-blocked dNTPs, reaction conditions for a deprotection step may comprise the following: 700 mM NaNCh; 1 M sodium acetate (adjusted with acetic acid to pH in the range of 4.8-6.5), where the deprotection reaction may be carried out in a 50 pL volume, at a temperature within the range of from room temperature to about 45°C for a duration of about 30 seconds to several minutes. Washes may be performed with the cacodylate buffer without the components of the coupling reaction (e.g. enzyme, monomer, divalent cations).

[0034] Depending on particular applications, the steps of deprotection and / or cleaving may include a variety of chemical or physical conditions, e.g. light, heat, pH, presence of specific reagents, such as enzymes, which are able to cleave a specified chemical bond. Guidance in selecting 3’-O-blocking groups and corresponding de-blocking conditions may be found in the following references, which are incorporated by reference: Benner, U.S. patents 7544794 and 8212020; U.S. patent 5808045; U.S. patent 8808988; International patent publication WO9 1 / 06678; and references cited below. In some embodiments, the cleaving agent (also sometimes referred to as a de-blocking reagent or agent) is a chemical cleaving agent, such as, for example, dithiothreitol (DTT). In alternative embodiments, a cleaving agent may be an enzymatic cleaving agent, such as, for example, a phosphatase, which may cleave a 3’- phosphate blocking group. It will be understood by the person skilled in the art that the selection of deprotection reagent depends on the type of 3 ’-nucleotide blocking group used, whether one or multiple blocking groups are being used, whether initiators are attached to living cells or organisms or to solid supports, and the like, that necessitate mild treatment. For example, a phosphine, such as tris(2-carboxyethyl)phosphine (TCEP) can be used to cleave a 3’0- azidomethyl group, palladium complexes or phosphonate compounds can be used to cleave a 3’O-allyl group. In particular embodiments, the cleaving reaction involves deprotecting a 3’0- amino blocking group by application of a phosphonate compound, in particular a carbonylbisphosphonate compound as described in International patent application PCT7EP2024 / 062042.

[0035] According to another embodiment, the reaction may start with a 3’-O-protected initiator nucleic acid. In such embodiments, the invention relates to a method of synthesizing a polynucleotide, wherein the method comprises the steps of providing a 3 ’-0 -protected initiator nucleic acid and(a) performing a cycle comprising the steps of:(i) deprotecting the 3'-O-protected elongated initiator nucleic acid to form an elongated nucleic acid having a free 3’ - hydroxyl,(ii) contacting a template-independent polymerase with an initiator nucleic acid and a 3’-O-protected nucleoside triphosphate, so that the initiator nucleic acid is elongated by the template-independent polymerase activity by incorporation of the 3’ -O-protected nucleoside triphosphate, to form a 3 '-O-protected elongated initiator nucleic acid,(b) repeating cycles of step (a) to further elongate the initiator nucleic acid, until the polynucleotide is synthesized,(c) wherein the template-independent polymerase is anchored to a solid support during cycles of step (a).In some embodiments, the method comprises a further step of deprotecting the 3’-O-protected synthesized polynucleotide.

[0036] Polynucleotide Products

[0037] Some aspects of the invention relate to a polynucleotide produced from a method as described herein. Some aspects of the invention relate to a polynucleotide produced by a template-independent polymerase as described herein. Some aspects of the invention relate to compositions comprising said polynucleotide products.

[0038] In some embodiments, the polynucleotide may be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) molecule or polymer, or in some cases may be a hybrid of DNA and RNA. In one or more embodiments, these may be referred to as polyribonucleic acids, or polydeoxyribonucleic acids. Alternatively, the polynucleotide may be an artificial polynucleotide or nucleic acid analogue selected from PNA, LNA, GNA, TNA, and HNA. In some embodiments the polynucleotide may be a DNA or RNA molecule or polymer. In some embodiments the polynucleotide may be single stranded (ss) or double stranded (ds). In some embodiments the polynucleotide may be selected from ssRNA, ssDNA, dsRNA, and dsDNA.

[0039] In some embodiments the polynucleotide product may be of any length. In some embodiments the polynucleotide product may be up to 1000 nucleotides in length, between 5 to 1000 nucleotides in length, between 5 to 900 nucleotides in length, between 5 to 800 nucleotides in length, between 5 to 700 nucleotides in length, between 5 to 600 nucleotides in length, between 10 to 500 nucleotides in length, between 10 to 400 nucleotides in length, between 10 to 300 nucleotides in length, between 10 to 200 nucleotides in length, between 10 to 100 nucleotides in length, between 10 to 50 nucleotides in length, between 10 to 40 nucleotides in length, between 10 to 30 nucleotides in length, between 10 to 20 nucleotides in length.

[0040] In one or more embodiments, the polynucleotide product may be up to 100 nucleotides in length, up to 90 nucleotides in length, up to 80 nucleotides in length, up to 70 nucleotides inlength, up to 60 nucleotides in length, up to 50 nucleotides in length, or up to 40 nucleotides in length.

[0041] In one or more embodiments, the polynucleotide product may comprise a high number of C or G nucleotides. In one or more embodiments, the polynucleotide may be classified as a ‘difficult’ sequence for the template-free polymerase variant to synthesize. In some embodiments, the polynucleotide may comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% C or G nucleotides. In some embodiments, the polynucleotide may comprise a majority of C or G nucleotides, suitably over 50% C or G nucleotides.

[0042] The polymerase may remain anchored to the solid support for at least 2 cycles, in particular at least 3, 4, 5, 6, 7, 8, 8 or 10 cycles of step (a). In particular, the polymerase remains anchored to the solid support for at least 15, 20, 25, 30, 35, 40, 45 or 50 cycles of step (a). More particularly, the polymerase remains anchored to the solid support for at least 75 or 100 cycles of step (a). Each cycle corresponds to the addition of one nucleoside triphosphate to the initiator nucleic acid being elongated.

[0043] The reaction may occur in any area, zone or volume suitable for a template-independent polymerase-driven nucleic acid extension reaction, e.g. within a reaction chamber or reaction compartment. The solid support may be fixed within the reaction chamber. Preferably, the polymerase is anchored within the reaction chamber.

[0044] The solid support is for instance selected from a bead, a planar surface, an array, a chip and a well. The solid support may be coated and / or functionalized, for attachment of the polymerase and / or initiator nucleic acid thereto.

[0045] In some embodiments, the polymerase may be anchored at a concentration of from 2.0 pM to 50 pM purified polymerase, preferably about 25 pM purified polymerase

[0046] The polymerase may be anchored in different ways to the solid support.

[0047] In some embodiments, the polymerase is anchored directly to the solid support, i.e. by direct attachment of the polymerase to the solid support. Alternatively, or in addition, the polymerase is immobilized to the solid support through attachment to an anchor, wherein the anchor is attached to the solid support. The anchor may be any structure or compound which serves as intermediate to anchor the polymerase to the solid support. The anchor is directly orindirectly attached to the solid support. In preferred embodiments, the anchor is a nucleic acid, preferably an initiator nucleic acid.

[0048] In some embodiments, the polymerase is attached to the initiator nucleic acid. Preferably, the initiator nucleic acid is anchored, in particular attached, to the solid support. Alternatively, the initiator nucleic acid may not be anchored, in particular attached to the solid support, e.g. may be in solution. Preferably, the polymerase is attached to the initiator nucleic acid and the initiator nucleic acid is attached to the solid support. The inventors have shown that such attachment of the polymerase to the initiator nucleic acid may anchor the polymerase to the solid support in a solid and stable manner. The solid support, initiator nucleic acid and polymerase form a complex which remains bound together during several cycles of initiator nucleic acid extension.

[0049] The polymerase may remain attached to the initiator nucleic acid for at least 2 cycles, in particular at least 3, 4, 5, 6, 7, 8, 9 or 10 cycles of step (a). In particular, the polymerase remains attached to the initiator nucleic acid for at least 15, 20, 25, 30, 35, 40, 45 or 50 cycles of step (a). More particularly, the polymerase remains attached to the initiator nucleic acid for at least 75 or 100 cycles of step (a). Each cycle corresponds to the addition of one nucleoside triphosphate to the initiator nucleic acid being elongated.

[0050] The initiator nucleic acid may be attached to the solid support by covalent or non- covalent attachment. In particular, the initiator nucleic acid is attached by its 5 ’-end and has a free 3 ’-end, available for extension by the template-independent polymerase. In some embodiments, the initiator nucleic acid is attached to the support by covalent attachment of its 5 ’-end to the surface of the support through a covalent linker. Attachment is preferably carried out by reacting a 5 ’-terminal NEE of the initiator nucleic acid with a functionalized surface of the solid support. In some embodiments, the initiator nucleic acid is attached to the solid support by covalent attachment to a functionalized surface of the solid support selected from a cyanogen bromide (CNBr)-functionalized surface, a N-hydroxysuccinimide (NHS)-functionalized surface and a glyoxal- functionalized surface.

[0051] In some embodiments, the initiator nucleic acid is attached to the solid surface by a linker comprising an amine bond, an amide bond or an isourea bond.

[0052] Preferably, the linker comprises a spacer between the support and the linker functionality. The spacer may be included to avoid steric hindrance during the linking process and / or during performance of the polynucleotide synthesis method. Typically, the spacer is ashort, flexible group, for instance an optionally substituted C1-C20 alkyl, optionally substituted C3-C20 heterocyclyl or an optionally substituted C5-20 aryl.

[0053] The solid support may comprise a polysaccharide polymer material, in particular a gelling polysaccharide polymer material. In some embodiments, the polysaccharide polymer material comprises agarose such as Sepharose®. More preferably, the solid support is an agarose bead.

[0054] In some embodiments, the polymerase and the initiator nucleic acid are attached by binding between at least one tag moiety comprised in or attached to the polymerase and at least one capture moiety comprised in or attached to the initiator nucleic acid. Accordingly, in some embodiments, the polymerase comprises, or is attached to, at least one tag moiety and the initiator nucleic acid comprises, or is attached to, at least one capture moiety, wherein the tag moiety and the capture moiety are capable of binding with each other, so as to attach the polymerase and the initiator nucleic acid. The binding of the tag moiety and the capture moiety may be covalent, e.g. it occurs by conjugation between two moieties which create a covalent bond in appropriate conjugation conditions. The binding of the tag moiety and the capture moiety may also be non-covalent, such as in an antibody-antigen interaction. In some embodiments, the tag moiety is covalently attached to the capture moiety.

[0055] In some embodiments, the initiator nucleic acid and the polymerase are in a molar ratio of from about 10: 1 to about 1 : 10, preferably from about 5: 1 to about 1 :5, still preferably from about 3:1 to about 1 :3, more preferably from about 2: 1 to 1 :2, for instance about 1 :2, 2: 1 or about 1 : 1. In some embodiments, the initiator nucleic acid and the polymerase are in a molar ratio of more than 1 :5, preferably more than 1 :4, more preferably more than 1 :3, still more preferably more than 1 :2.

[0056] The tag moiety may be a polypeptide moiety. In such case, it may be expressed in fusion with the polymerase. In some embodiments, the polymerase and the tag moiety may thus form a fusion protein. The fusion protein may comprise a polymerase moiety with templateindependent polymerase activity and a tag moiety for attaching the polymerase to an initiator nucleic acid.

[0057] There are multiple ways of coupling the polymerase and the initiator nucleic acid, including conjugation between a streptavidin, avidin, neutravidin, captavidin, traptavidin, or a biotin-binding antibody and a biotin or desthiobiotin or their derivatives, and conjugation through a self-labelling protein.

[0058] In some embodiments, the polymerase and the initiator nucleic acid are attached by conjugation between a biotin-binding moiety, such as streptavidin, avidin, neutravidin, captavidin, traptavidin, or a biotin-binding antibody or a derivative or fragment thereof, and a biotin or a derivative thereof. In one embodiment, the tag moiety comprises a biotin-binding moiety and the capture moiety comprises a biotin or a derivative thereof. In another embodiment, the tag moiety comprises a biotin or a derivative thereof and the capture moiety comprises a biotin-binding moiety. Any biotin-binding moiety which can bind biotin is herein encompassed, in particular streptavidin, avidin, neutravidin, captavidin, traptavidin and biotinbinding antibodies or their derivatives.

[0059] In some embodiments, the polymerase and the initiator nucleic acid are attached by conjugation between at least one self-labelling protein and at least one nucleotide or nucleotide analogue, preferably a deoxyuridine. In preferred embodiments, the self-labelling protein is comprised in or attached to the polymerase and the nucleotide analogue is comprised in or attached to the initiator nucleic acid.

[0060] In some embodiments, the tag moiety is an uracil DNA glycosylase, preferably a UDGx enzyme. More preferably, the uracil DNA glycosylase comprises an amino acid sequence as set forth in any one of SEQ ID NO: 2 to 13, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with any one of the sequences set forth in SEQ ID NO: 2 to 13, or a biologically active fragment thereof. In some embodiments, the uracil DNA glycosylase may comprise 1 to 20, in particular 1 to 10, more particularly 1 to 5, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes with respect to any one of the sequences set forth in SEQ ID NO: 2 to 13.

[0061] In some embodiments, the uracil DNA glycosylase comprises an amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with any one of the sequences set forth in SEQ ID NO:7, or a biologically active fragment thereof. In some embodiments, the uracil DNA glycosylase may comprise 1 to 20, in particular 1 to 10, more particularly 1 to 5, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes with respect to any one of the sequences set forth in SEQ ID NO:7.

[0062] In some embodiments, the uracil DNA glycosylase comprises an amino acid sequence according to SEQ ID NO:3, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 3, or a biologically active fragment thereof, preferablywherein the amino acid sequence comprises mutations at one or more or all of the following positions: A12, V23, A25, V33, A38, T46, V50, M51, R59, T62, Q76, D80, A81, E88, T99, R104, L106, S 114, D115, L137, K144, L153, G166, G171, L172, G173, L177, G193, and E207, or corresponding positions thereto.

[0063] In some embodiments the amino acid sequence comprises one or more or all of the following mutations: A12E, V23A, A25G, V33R, A38T, T46S, V50M, M51L, R59Q, T62R, Q76D, D80E, A81E, E88Q, T99K, R104K, L106R, S114T, D115E, L137C, K144Q, L153V, G166E, G171T, LI 72V, G173D, L177R, G193E, and E207R or each of said mutations at corresponding positions thereto.

[0064] In some embodiments, the uracil DNA glycosylase comprises an amino acid sequence according to SEQ ID NO:8, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 8, or a biologically active fragment thereof preferably wherein the amino acid sequence comprises mutations at one or more or all of the following positions: All, G19, M31, M92, E95, Q139, A155, T156, T161, S171, T172, H176, T178, L183, V203, E205, A206, A216, R220, and G222 or mutations at corresponding positions thereto.

[0065] In some embodiments the amino acid sequence comprises one or more or all of the following mutations: A11D, G19D, M31R, M92I, E95V, Q139E, A155S, T156D, T161A, S171A, T172S, H176D, T178A, L183V, V203A, E205Q, A206S, A216G, R220G, and G222A or each of said mutations at corresponding positions thereto.

[0066] In some embodiments, the uracil DNA glycosylase comprises an amino acid sequence according to SEQ ID NO: 10, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 10, or a biologically active fragment thereof, preferably wherein the amino acid sequence comprises mutations at one or more or all of the following positions: G10, T17, S27, N37, E39, R40, L52, V53, G90, E91, E104, A107, A108, G110, Al 18, G120, 1166, P169, D170, 1173, P174, A178, 1183, 1188, Q195, E197, L200, and G202, or corresponding positions thereto.

[0067] In some embodiments the amino acid sequence comprises one or more or all of the following mutations: G10A, T17R, S27R, N37D, E39T, R40Q, L52M, V53M, G90E, E91R, E104T, A107E, A108G, G110K, A118S, G120T, I166V, P169L, D170P, I173V, P174E, A178R, I183V, I188V, Q195D, E197D, L200F, and G202A, or each of said mutations at corresponding positions thereto.

[0068] In some embodiments, the capture moiety is a deoxyuridine.

[0069] The polymerase may be or may comprise any template-independent polymerase, in particular a TdT, more particularly a TdT variant. The TdT or TdT variant can be from any species or be a chimeric protein. By “chimeric protein”, is meant that portions of the variant are from at least 2 different species. Said chimeric protein is formed by the addition, and in particular fusion or conjugation, of one or more predetermined sequences of a protein of one species and at least one another predetermined sequence of a second species which is a member of the polX family, in particular a TdT. Preferably, the TdT variant is a chimeric protein, more preferably the chimeric protein comprises portions from 2 different species, in particular a predetermined sequence from mouse and a predetermined sequence from bovine.

[0070] In some embodiments, the TdT variant comprises an amino acid sequence as set forth in SEQ ID NO: 17 to 50 or an amino acid sequence having at least 70%, preferably 80%, more preferably 85%, even more preferably 90%, 95%, 98% or 99% identity with respect to any one of the sequences set forth in SEQ ID NO: 17 to 50, or a biologically active fragment thereof.

[0071] In some embodiments, the TdT variant comprises 1 to 20, in particular 1 to 10, more particularly 1 to 5, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes with respect to the sequence set forth in any one of SEQ ID NO: 17 to 50.

[0072] In some embodiments , the TdT variant does not comprise the N-terminal part of the corresponding wild-type TdT. In particular, TdT variants according to the invention are N- terminally truncated TdTs lacking amino acids residues 1 to 147 of the corresponding wild-type parent NP 001036693.1 [Mus musculus] TdT sequence. In some embodiments, the N-terminal part of the truncated TdT variants according to the invention may nevertheless comprise an inserted Tag, such as for example the Tag of SEQ ID NO: 51 added at the N-terminus of the variant.

[0073] In some embodiments, the TdT variant is as described in any one of WO2017 / 216472, W02019 / 135007, W02020 / 099451, WO2021 / 116270, WO2021 / 213903, WO2022 / 063835, WO2023083997, WO2020 / 239737 and WO2023 / 083999, the entire content of which is incorporated by reference

[0074] In some embodiments, the polymerase is a fusion protein comprising a moiety with template-independent polymerase activity and a self-labelling protein for attaching thepolymerase to an initiator nucleic acid protein. In some embodiments, the self-labelling protein has uracil DNAglycosylase activity. In some embodiments, the tag moiety comprises an UDGx enzyme or a variant thereof, in particular any one of UdGxl, UdGx2, UdGx3, UdGx4, UdGx5, UdGx6, UdGx7, UdGx8, UdGx9 or UdGxlO or any variant, biologically active fragment or functional equivalent thereof.

[0075] In some embodiments, the moiety with template-independent polymerase activity has terminal deoxynucleotidyl transferase (TdT) activity. In some embodiments the polymerase is a fusion protein comprising a TdT moiety and an UDGx moiety.

[0076] In some embodiments, the moiety with template-independent polymerase activity and the tag moiety are linked by a peptide linker. In some embodiments, the peptide linker comprises from 4 to 50 amino acids, in particular from 5 to 25 amino acids, more particularly from 6 to 20 amino acids.

[0077] In some embodiments, the fusion protein comprises a moiety with template-independent polymerase activity, comprising the amino acid sequence set forth in any one of SEQ ID NO: 17 to 50 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with respect to the sequence set forth in any one of SEQ ID NO: 17 to 50, or a biologically active fragment thereof, and a tag moiety for attaching the polymerase, wherein said tag moiety comprises an amino acid sequence as set forth in any one of SEQ ID NO: 2 to 13 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with a sequence set forth in any one of SEQ ID NO: 2 to 13, or a biologically active fragment thereof.

[0078] In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with respect to the sequence set forth in SEQ ID NO: 1 or a biologically active fragment thereof. In some embodiments, the fusion protein comprises 1 to 20, in particular 1 to 10, more particularly 1 to 5, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes with respect to the sequence set forth in SEQ ID NO: 1.

[0079] In some embodiments, the initiator nucleic acid comprises at least one deoxyuridine. Deoxyuridine can conjugate with enzymes such as UDGx, anchoring the polymerase to the initiator nucleic acid.

[0080] In some embodiments, the polymerase is attached to the solid support, i.e. it is anchored by direct attachment to the solid support. In particular, the polymerase may be attached to the solid support and not attached to the initiator nucleic acid as an intermediate anchor. The polymerase may be attached covalently or non-covalently to the solid support. In some embodiments, the polymerase is attached to the solid support by covalent attachment to a functionalized surface of the solid support selected from a cyanogen bromide (CNBr)- functionalized surface, a N-hydroxysuccinimide (NHS)-functionalized surface and a glyoxal- functionalized surface.In some embodiments, the polymerase is attached to the solid surface by a linker comprising an amine bond, an amide bond or an isourea bond. Preferably, the linker comprises a spacer between the support and the linker functionality. The spacer may be included to avoid steric hindrance during the linking process and / or during performance of the polynucleotide synthesis method. Typically, the spacer is a short, flexible group, for instance an optionally substituted C1-C20 alkyl, optionally substituted C3-C20 heterocyclyl or an optionally substituted C5-20 aryl.

[0081] In some embodiments, the polymerase is attached to the solid support by conjugation between a biotin-binding moiety, such as streptavidin, avidin, neutravidin, captavidin, traptavidin, and biotin-binding antibodies or a derivative or fragment thereof, and a biotin or a derivative thereof. In one embodiment, the polymerase comprises or is attached to a biotinbinding moiety and the solid support comprises or is attached to a biotin or a derivative thereof. In another embodiment, the polymerase comprises or is attached to a biotin or a derivative thereof and the solid support comprises or is attached to a biotin-binding moiety. Any biotinbinding moiety which can bind biotin is herein encompassed, in particular streptavidin, avidin, neutravidin, captavidin, traptavidin, and biotin-binding antibodies or their derivatives.In embodiments in which the polymerase is attached to the solid support, in particular directly attached to the solid support without being attached to the initiator nucleic acid as anchor, the initiator nucleic acid may also be attached to the solid support. In some embodiments, the initiator nucleic acid is attached to the support by covalent attachment of its 5 ’ -end to the surface of the support through a covalent linker. Linkers for attaching the initiator nucleic acid to the solid support are described in the present specification and applicable to the present embodiment.

[0082] In some embodiments, the initiator nucleic acid is attached to the solid support by conjugation between a biotin-binding moiety, such as streptavidin, avidin, neutravidin,captavidin, traptavidin, and biotin-binding antibodies or a derivative or fragment thereof, and a biotin or a derivative thereof. In one embodiment, the initiator nucleic acid comprises or is attached to a biotin-binding moiety and the solid support comprises or is attached to a biotin or a derivative thereof. In another embodiment, the initiator nucleic acid comprises or is attached to a biotin or a derivative thereof and the solid support comprises or is attached to a biotinbinding moiety. Any biotin-binding moiety which can bind biotin is herein encompassed, in particular streptavidin, avidin, neutravidin, captavidin, traptavidin, and biotin-binding antibodies or their derivatives.

[0083] In some embodiments, the polymerase and the initiator nucleic acid are both attached onto the solid support. In particular, the polymerase and the initiator nucleic acid may be both attached independently onto the solid support, each at a different attachment point on the solid support. More particularly, the polymerase and the initiator nucleic acid are both attached to the solid support, at a sufficient proximity from each other, for the polymerase to extend the initiator nucleic acid during cycles (a) of elongation.

[0084] In some embodiments, the method comprises, before step (a), a step of providing a solid support. Preferably, the solid support is provided within a reaction chamber. Any type of solid support can be provided, as described in the present specification.

[0085] In some case, the method comprises, before step (a), a step of providing a solid support comprising a template-independent polymerase anchored to the solid support.

[0086] In some embodiments, the method comprises, before step (a), a step of immobilizing a template-independent polymerase to a solid support. Immobilization may be carried out through the immobilization modalities described in the present specification.

[0087] In some embodiments, the method comprises, before step (a), a step of providing a solid support. Preferably, the solid support is provided within a reaction chamber. Any type of solid support can be provided, as described in the present specification.

[0088] In some embodiments, step (ii) includes contacting the 3'-O-protected elongated initiator nucleic acid with at least one deprotecting reagent. In some embodiments, the deprotection reagent is a phosphonate compound, in particular a carbonylbisphosphonate compound phosphonate compound having the formula R-P(=0)(0M)0M in which: each M is independently selected from the group consisting of: H; a monovalent or divalent metal atom; HNR63+or NR64+ wherein each R6independently designates Hor a linear or branched alkyl group having from 1 to 6 carbon atoms; a protonated organic base; a linear or branched alkyl group having from 1 to 6 carbon atoms ; and a Si(R4)s group wherein each R4 is independently selected from an aryl group and a linear or branched alkyl group having from 1 to 6 carbon atoms; andR is -CO-Ri wherein Ri is selected from: (i) a linear or branched alkyl group having from 1 to 6 carbon atoms, (ii) an aryl group and (iii) a -P(=0)(0M)0M group.

[0089] Examples of phosphonate compounds are described in International patent application PCT / EP2024 / 062042, the content of which is incorporated by reference in its entirety. Preferably, 3’0-amino blocking group is deprotected by application of a carbonylbisphosphonate compound.

[0090] In some embodiments, step (a) of the method comprises one or more washing steps. Washing steps serve to remove reagents from the solid support and / or from the reaction chamber, such as unincorporated 3’-O-protected nucleoside triphosphates and / or deprotection reagents. In particular, step (a) may comprise removing unincorporated 3 ’ -O-protected nucleoside triphosphates. Such removal can occur after step (i), i.e. between step (i) and step (ii) and / or after step (ii). Step (a) may comprise more than one washing steps. In particular, step (a) may comprise a washing step after step (i) to remove unincorporated 3 ’ -O-protected nucleoside triphosphates and a washing step after step (ii) to remove the deprotection reagent(s). An advantage of the invention is that, due to the immobilization of the polymerase, the polymerase is not washed during the washing steps, i.e. it remains anchored to the solid support. It thus remains available and active for incorporating further nucleotides to the initiator nucleic acid, in subsequent cycles of step (a).

[0091] In some embodiments, the method further comprises steps of purifying the synthesized polynucleotide and / or collecting the synthesized polynucleotide.

[0092] In some embodiments, the template-independent polymerase is a terminal deoxynucleotidyl transferase (TdT) or a variant thereof (e.g. as described in any one of WO20 17 / 216472, W02019 / 135007, W02020 / 099451, WO2021 / 116270, WO2021 / 213903, WO2022 / 063835, WO2023083997, WO2020 / 239737 and WO2023 / 083999).

[0093] In some cases, no further template-independent polymerase is added for at least two consecutive cycles of elongation (a). In some cases, no further template-independent polymerase is added during cycles of elongation (a), preferably until the polynucleotide issynthesized. The anchored template-independent polymerase is thus preferably the only template-independent polymerase used in the performance of the method.

[0094] In some embodiments, the method comprises a further step of detaching the synthesized polynucleotide from the solid support. For instance, the initiator nucleic acid includes a cleavable group and the synthesized polynucleotide is detached from the solid support by cleavage of the cleavable group. In some embodiments, the cleavable group is an enzymatically cleavable group selected from a deoxyuridine (dU), a deoxyinosine (di), an inosine, an uracil, a nitroindole, a hypoxanthine, a thymidine glycol, a 5 -hydroxyuracil, 5,6-dihydrouracil and a 5-hydroxy cytosine.

[0095] In some embodiments, the method of the invention is a massively parallel method for synthesizing a plurality of polynucleotides. The method may comprise the steps of:(a) performing a cycle comprising the steps of:(i) contacting a template-independent polymerase with an initiator nucleic acid and a 3’- O-protected nucleoside triphosphate, so that the initiator nucleic acid is elongated by the template-independent polymerase activity by incorporation of the 3’ -O-protected nucleoside triphosphate, to form a 3 '-O-protected elongated initiator nucleic acid,(ii) deprotecting the 3 '-O-protected elongated initiator nucleic acid to form an elongated nucleic acid having a free 3’ - hydroxyl,(b) repeating cycles of step (a) to further elongate the initiator nucleic acid, until the polynucleotide is synthesized, wherein the template-independent polymerase is anchored to a solid support during cycles of step (a).

[0096] Any of the characteristics described in the specification in relation with the method of the invention, in particular regarding the template independent polymerase, the initiator nucleic acid and / or the solid support, is applicable to the other aspects of the invention described in the present specification, in particular massively parallel methods for synthesizing a plurality of polynucleotides or the following aspects, such as those relating to a template-independent polymerase, a nucleic acid molecule, a solid support, a kit and a use.

[0097] In another aspect, the invention relates to a template-independent polymerase for use in the method of the invention, wherein said polymerase comprises a moiety with template-independent polymerase activity and a tag moiety, in particular wherein the tag moiety is adapted to attach the polymerase to an initiator nucleic acid, wherein said initiator nucleic acid is a substrate for the template-independent polymerase.

[0098] The template-independent polymerase may have any of the features or combination of features described in the present specification, in relation to the method of the invention.

[0099] The moiety with template-independent polymerase activity may be or comprise any template-independent polymerase, in particular a TdT, more particularly a TdT variant. The TdT or TdT variant can be from any species or be a chimeric protein. By “chimeric protein”, is meant that portions of the variant are from at least 2 different species. Said chimeric protein is formed by the addition, and in particular fusion or conjugation, of one or more predetermined sequences of a protein of one species and at least one another predetermined sequence of a second species which is a member of the polX family, in particular a TdT. Preferably, the TdT variant is a chimeric protein, more preferably the chimeric protein comprises portions from 2 different species, in particular a predetermined sequence from mouse and a predetermined sequence from bovine.

[0100] In some embodiments, the TdT variant comprises an amino acid sequence as set forth in SEQ ID NO: 17 to 50 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with respect to a sequence set forth in any one of SEQ ID NO: 17 to 50.

[0101] In some embodiments, the TdT variant comprises 1 to 20, in particular 1 to 10, more particularly 1 to 5, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes with respect to an amino acid sequence set forth in any one of SEQ ID NO: 17 to 50.

[0102] In some embodiments , the TdT variant does not comprise the N-terminal part of the corresponding wild-type TdT. In particular, TdT variants according to the invention are N-terminally truncated TdTs lacking amino acids residues 1 to 147 of the corresponding wildtype parent NP 001036693.1 [Mus musculus] TdT sequence. In some embodiments, the N- terminal part of the truncated TdT variants according to the invention may nevertheless comprise an inserted Tag, such as for example the Tag of SEQ ID NO: 51 added at the N- terminus of the variant.

[0103] In some embodiments, the TdT variant is as described in any one of WO20 17 / 216472, W02019 / 135007, W02020 / 099451, WO2021 / 116270, WO2021 / 213903, WO2022 / 063835, WO2023083997, WO2020 / 239737 and WO2023 / 083999, the entire content of which is incorporated by reference

[0104] In some embodiments, the polymerase can remain attached to the initiator nucleic acid during at least 2 cycles of polymerase-mediated elongation of the initiator nucleic acid, in particular at least 3, 4, 5, 6, 7, 8, 8 or 10 cycles of nucleic acid extension. In particular, the polymerase can remain attached to the nucleic acid for at least 15, 20, 25, 30, 35, 40, 45 or 50 cycles of nucleic acid extension.

[0105] In some embodiments, the polymerase can be attached to the initiator nucleic acid through covalent or non-covalent attachment between the tag moiety and a capture moiety included in the initiator nucleic acid. In some embodiments, the tag moiety and / or capture moiety remains unmodified upon elongation of the initiator nucleic acid by the polymerase. Preferably, the attachment of the polymerase to the initiator nucleic acid is stable for at least 2 cycles of elongation, preferably 3, 4, 5, 6 7, 8, 9 or 10 cycles of elongation, more preferably 15, 20, 25, 30, 35, 40, 45 and 50 cycles of elongation. In particular, said tag moiety and / or capture moiety is not incorporated in the initiator nucleic acid by the polymerase, e.g. it is not a nucleoside or a part of a nucleoside or does not comprise a nucleoside.

[0106] In some embodiments, said tag moiety is selected from a biotin-binding moiety, such as streptavidin, avidin, neutravidin, captavidin, traptavidin, biotin-binding antibodies or a derivative or fragment thereof, a biotin or derivative thereof, and a self-labelling protein. In some embodiments, the tag moiety is an uracil DNA glycosylase, preferably UDGx.

[0107] In some embodiments, the tag moiety is an uracil DNA glycosylase, preferably a UDGx enzyme, and the nucleotide analogue is a deoxyuridine. More preferably, the uracil DNA glycosylase comprises an amino acid sequence set forth in any one of SEQ ID NO: 2 to13, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with a sequence set forth in any one of SEQ ID NO: 2 to 13, or a biologically active fragment thereof. In some embodiments, the uracil DNA glycosylase may comprise 1 to 20, in particular 1 to 10, more particularly 1 to 5, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 amino acid changes with respect a sequence set forth in any one of SEQ ID NO: 2 to 13. In some embodiments, the uracil DNA glycosylase is as described in the various aspects of the invention.

[0108] In some embodiments, the moiety with template-independent polymerase activity and the tag moiety are linked by a peptide linker. In some embodiments, the peptide linker comprises from 4 to 50 amino acids, in particular from 5 to 25 amino acids, more particularly from 6 to 20 amino acids.

[0109] In some embodiments, the fusion protein comprises a moiety with templateindependent polymerase activity, comprising an amino acid sequence set forth in any one of SEQ ID NO: 17 to 50, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with respect to a sequence set forth in any one of SEQ ID NO: 17 to 50, or a biologically active fragment thereof, and a tag moiety for attaching the polymerase, comprising the amino acid sequence set forth in any one of SEQ ID NO: 2 to 13 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with a sequence set forth in any one of SEQ ID NO: 2 to 13, or a biologically active fragment thereof,.

[0110] In some embodiments, the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with respect to the sequence set forth in SEQ ID NO: 1, or a biologically active fragment thereof.

[0111] In some embodiments, the polymerase comprises an anchor moiety for attaching the polymerase to a solid support. In particular, said anchor moiety is a covalent linker selected from a thiophosphate linker, a thiol linker, an amino-containing linker and a silane linker, or wherein said anchor moiety is a streptavidin-binding moiety such as a streptavidin-binding polypeptide, e.g. biotin, desthiobiotin and their derivatives.

[0112] In some embodiments, the polymerase is a fusion protein comprising a moiety with template-independent polymerase activity and a tag moiety for attaching the polymerase to an initiator nucleic acid. In some embodiments, the tag moiety is a self-labelling protein. In some embodiments, the tag has uracil DNA glycosylase activity. In some embodiments, the tag moiety comprises an UDGx enzyme or a variant thereof.

[0113] In some embodiments, the moiety with template-independent polymerase activity has terminal deoxynucleotidyl transferase (TdT) activity. In some embodiments the polymerase is a fusion protein comprising a TdT moiety and an UDGx moiety.

[0114] In some aspects, the invention relates to an initiator nucleic acid for use in the method of the invention, wherein said initiator nucleic acid includes a capture moiety for attaching the nucleic acid molecule to a template-independent polymerase.

[0115] In some embodiments, the capture moiety is selected from a biotin or a derivative thereof, a biotin-binding moiety, such as streptavidin, avidin, neutravidin, captavidin, traptavidin, and a biotin-binding antibody or a derivative or fragment thereof, and a nucleotide or a nucleotide analogue. In particular, the nucleotide analogue is a deoxyuridine.

[0116] In some embodiments, said initiator nucleic acid includes an anchor moiety, preferably at its 5 ’-end, for attaching the initiator nucleic acid to a support. In some embodiments, the initiator nucleic acid has a free 3 ’-hydroxyl end.

[0117] In some embodiments, the initiator nucleic acid comprises a cleavable group, preferably an enzymatically cleavable group selected from a deoxyuridine (dU), a deoxyinosine (di), an inosine, a uracil, a nitroindole, a hypoxanthine, a thymidine glycol, a 5 -hydroxyuracil, 5,6-dihydrouracil and a 5 -hydroxy cytosine.

[0118] In some aspects, the invention relates to a conjugate comprising a polymerase, preferably a template-independent polymerase, conjugated to an initiator nucleic acid. The conjugate may have any of the characteristics described herein. In particular, the polymerase may have any of the characteristics described herein, the initiator nucleic acid may have any of the characteristics described herein, and the conjugation between the polymerase and the initiator nucleic acid may have any of the characteristics described herein.

[0119] In some aspects, the invention relates to a solid support, in particular a functionalized solid support, for synthesis of a polynucleotide, wherein the solid support comprises, anchored to the solid support: at least one template-independent polymerase; and optionally at least one initiator nucleic acid.In some embodiments, the template-independent polymerase and / or initiator nucleic acid are as described in the present specification. In some embodiments, the template-independent polymerase is a terminal deoxy transferase (TdT). In some embodiments, the templateindependent polymerase is indirectly attached to the solid support. In particular, the templateindependent polymerase is immobilized to the solid support through attachment to the initiator nucleic acid, itself attached to the solid support. In such embodiment, the template-independent1 polymerase is attached to the initiator nucleic acid and the initiator nucleic acid is attached to the solid support. Such attachment of the template-independent polymerase to the initiator nucleic acid immobilizes the polymerase to the solid support. In some embodiments, the solid support, the initiator nucleic acid and the template-independent polymerase preferentially form a complex which remains bound together during several cycles of initiator nucleic acid extension.

[0120] In some embodiments, the invention relates to a kit for performing the method of the invention, comprising: at least one template-independent polymerase; and at least one initiator nucleic acid.

[0121] In some embodiments, the template-independent polymerase and / or initiator nucleic acid are as described in the present specification

[0122] In particular, the kit may comprise: at least one template-independent polymerase, wherein said polymerase comprises a moiety with template-independent polymerase activity and a tag moiety; and at least one initiator nucleic acid, wherein said nucleic acid molecule includes a capture moiety; wherein the tag moiety of the template-independent polymerase binds to the capture moiety of the initiator nucleic acid.

[0123] In some embodiments, the kit comprises a solid support. In some embodiments, the template-independent polymerase and / or the initiator nucleic acid are attached or are attachable to the solid support.

[0124] In some embodiments, the kit further comprises one or more of:3’-O-protected nucleoside triphosphates; one or more reagent and / or buffer for a template-independent driven elongation reaction; one or more deprotection reagent and / or buffer; and a notice of instructions.In some aspects, the invention further relates to a device for performing the method of the invention, wherein said device comprises: a reaction chamber comprising a solid support; and means for dispensing buffers, reagents and solvents to the reaction chamber, wherein at least one template-free independent polymerase is anchored to the solid support.In some aspects, at least one initiator nucleic acid is anchored to the solid support.

[0125] Any of the features or combination of the features described in connection with the above aspects of the invention are applicable to the device of the invention.

[0126] In some aspects, the invention relates to the use of the polymerase of the invention, the nucleic acid molecule of the invention, the solid support of the invention, the kit of any one of the invention, and / or the device of the invention, for synthesizing one or more polynucleotides, in particular in a template-independent enzymatic synthesis method.

[0127] While the invention is amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. 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.Example 1

[0128] A fusion protein UDGx-TdT was produced using the following construct:[Histag]-SSA-[UDGx6]-[KETAAAKFERQHMDSE(RNAseA peptide linker)]-TdT variant, the sequence of which is set forth in SEQ ID NO: 1

[0129] The fusion protein was expressed in the commercial E. coli strains BL21 (DE3) (Novagen). The colonies that were capable of growing in kanamycin petri dishes were isolated. The fusion protein was expressed in 100 mL flasks with the addition of 50 pM of FeCh to the 2xYT medium and purified using Ni-NTA chromatography as described in US 2020 / 0002690. Protein amounts were determined by absorbance at 280 nm.

[0130] Initiator DNA (iDNA) sepharose resin was prepared by chemical conjugation of iDNA oligonucleotides on Cyanogen bromide-activated-Sepharose beads (CNBr Sepharose beads). The iDNA was the following dU-containing-iDNA oligonucleotide / 5AmMC12 / CCCCCCCCCCCCCCCTTT / ideoxyU / TTTTTT / ideoxyl / T

[0131] Subsequently, the fusion protein was conjugated to the iDNA as follows: the iDNA-loaded resin was washed on the column with the following buffer: IM NaCl, 25mM Tris pH 8.5, lOmM Imidazole (hereinafter, WB) and mixed with 2x of the UDGx TdT fusion protein in comparison to the concentration of iDNA in the resin). After Ihr of shaking at room temperature, the resin was washed with WB. The presence of the fusion protein was confirmed after 3 weeks at 4°C.

[0132] Using the resin thus prepared, enzymatic DNA Synthesis was performed with the following conditions for the Elongation Step: 47°C for 4 mins in the presence of 3’-0 amino protected dNTPs at a concentration of 500 pM. The deblocking step was carried out using a Deblocking Buffer comprising a carbonyl bisphosphonate deblocking agent used at a concentration of 20mM.Results

[0133] +1 end-labelling was performed to test the addition of 2 to 7 T (thymine) nucleotides, without further addition of TdT during the synthesis. Fig. 2 shows that the polyT addition is performed functionally by the immobilized TdT polymerase.Example 2

[0134] A synthesis of 52 mers was performed in the same synthesis conditions as those used in Example 1, except that the loading of the resin slurry was varied. Lanes 1 to 4 show the control resin without immobilized polymerase loaded between 200 pmoles and 750 pmoles of iDNA per reaction well with a concentration around 160 nmol / mL (1 : 750 pmoles / well; 2: 500pmoles / well; 3: 375 pmoles / well; 4: 250 pmoles / well). Lanes 5 to 8 show the resin according to the invention comprising UDGx-TdT, loaded between 15pL and 80pL with about 3pmoles iDNA per pL (5: 30 pl; 6: 50 pl; 7: 80 pl; 8: 15 pl). Slurry was -30% bead volume.

[0135] The synthesized sequence was as set forth in SEQ ID NO: 5.

[0136] Fig. 3 shows that synthesis was performed functionally by the immobilized TdT polymerase, demonstrating that the synthesis of a oligonucleotide around 50nt is possible with immobilized TdT, without further addition of polymerase during the cycles of extension. Fig. 4 shows that NGS-measured error rates with immobilized polymerase (1 : 15pl; 2: 30 pl) are comparable to control conditions, with a non-immobilized polymerase (3: 200 pmoles; 4: 375pmoles). This experiment shows that, even in non-optimized conditions for TdT immobilization, the TdT surprisingly shows robustness and high fidelity for DNA synthesis. Error rates, e.g. for deletion, are expected to be further reduced by optimization of the reaction conditions.

Claims

CLAIMS

1. A method of synthesizing a polynucleotide, wherein the method comprises the steps of:(a) performing a cycle comprising the steps of:(i) contacting a template-independent polymerase with an initiator nucleic acid and a 3 ’-0 -protected nucleoside triphosphate, so that the initiator nucleic acid is elongated by the template-independent polymerase activity by incorporation of the 3’ -O-protected nucleoside triphosphate, to form a 3 '-O-protected elongated initiator nucleic acid,(ii) deprotecting the 3 '-O-protected elongated initiator nucleic acid to form an elongated nucleic acid having a free 3’ - hydroxyl,(b) repeating cycles of step (a) to further elongate the initiator nucleic acid, until the polynucleotide is synthesized, wherein the template-independent polymerase is anchored to a solid support and / or attached to the initiator nucleic acid during cycles of step (a).

2. The method of claim 1, wherein the template-independent polymerase is attached to the initiator nucleic acid and the initiator nucleic acid is attached to the solid support, thereby anchoring the template-independent polymerase to the solid support.

3. The method of claim 1 or 2, wherein the template-independent polymerase and the initiator nucleic acid are attached by covalent attachment.

4. The method of any one of claims 1 to 3, wherein the template-independent polymerase and the initiator nucleic acid are attached by conjugation between at least one tag moiety comprised in and / or attached to the template-independent polymerase and at least one capture moiety comprised in and / or attached to the initiator nucleic acid.

5. The method of claim 4, wherein the template-independent polymerase and the initiator nucleic acid are attached by conjugation between at least one biotin-binding moiety and at least one biotin or a derivative thereof.

6. The method of any one of claims 1 to 5, wherein the template-independent polymerase is a fusion protein comprising a polymerase moiety with template-independent polymerase activity and a tag moiety for attaching the polymerase to the initiator nucleic acid.

7. The method of claim 6, wherein the tag moiety is a self-labelling protein.

8. The method of claim 6 or 7, wherein the tag moiety has uracil DNA glycosylase activity.

9. The method of claim 8, wherein the tag moiety is or comprises uracil DNA glycosylase X (UDGx) enzyme or a biologically active fragment thereof.

10. The method of any one of claims 1 to 9, wherein the template-independent polymerase is a fusion protein comprising a terminal deoxynucleotidyl transferase (TdT) moiety and an UDGx moiety.

11. The method of any one of claims 1 to 10, wherein the template-independent polymerase and / or initiator nucleic acid are attached to the solid support by covalent attachment.

12. The method of claim 11, wherein the template-independent polymerase and / or initiator nucleic acid are attached to the solid support by a linker comprising an amine bond, an amide bond or an isourea bond.

13. The method of any one of claims 1 to 10, wherein the template-independent polymerase and / or initiator nucleic acid are attached to the solid support by non-covalent attachment.

14. The method of claim 13, wherein the template-independent polymerase and / or initiator nucleic acid are attached to the solid support by non-covalent attachment between a streptavidin-binding moiety and streptavidin or a derivative thereof.

15. A template-independent polymerase for use in the method of the invention, wherein said template-independent polymerase includes a moiety with template-independent polymerase activity and a tag moiety for attaching the polymerase to an initiator nucleic acid.

16. The polymerase of claim 15, wherein said tag moiety is a biotin-binding moiety, such as streptavidin, avidin, neutravidin, captavidin, traptavidin, or a biotin-binding antibody or a derivative or fragment thereof, or a self-labelling protein.

17. The polymerase of claim 16, wherein the self-labelling protein is an uracil DNA glycosylase.

18. The polymerase of claim 17, wherein the uracil DNA glycosylase is UDGx.

19. The polymerase of claim 18, which is a fusion protein comprising a TdT moiety and an UDGx moiety.

20. A solid support for synthesis of a polynucleotide, wherein the solid support comprises, anchored to the solid support: at least one template-independent polymerase, in particular wherein the polymerase is according to any one of claims 15 to 19; and optionally at least one initiator nucleic acid, for use as substrate of the template-independent polymerase.

21. A kit for performing the method any one of claims 1 to 14, comprising: a template-independent polymerase, wherein said polymerase comprises a moiety with template-independent polymerase activity and a tag moiety for attaching the polymerase to an initiator nucleic acid; and an initiator nucleic acid, wherein said initiator nucleic acid comprises a capture moiety for attaching the nucleic acid molecule to the template-independent polymerase; wherein the tag moiety of the polymerase binds to the capture moiety of the nucleic acid molecule.

22. A device for performing the method of any one of claims 1 to 14, wherein said device comprises: a reaction chamber comprising a solid support to which a template-independent polymerase is anchored; and means for dispensing buffers, reagents and solvents to the reaction chamber.