Filtering device and method for polynucleotide enzymatic synthesis

Abstract

The present invention is directed to methods and device for template-free enzymatic synthesis of polynucleotides using a filtering barrier.

Description

[0001] FILTERING DEVICE AND METHOD FOR POLYNUCLEOTIDE

[0002] ENZYMATIC SYNTHESIS

[0003] BACKGROUND OF THE INVENTION

[0004] [1] The invention relates to the synthesis of biomolecules, and more particularly to the synthesis of RNA and DNA using filtration means.

[0005] [2] RNA and DNA polynucleotides are linear polymers of nucleotide monomers or analogues thereof which are capable of specifically binding to other polynucleotides by way of a regular pattern of monomer-to-monomer interactions. Polynucleotides typically range in size from a few monomeric units to several thousand monomeric units. Usually, polynucleotides comprise the four natural nucleotides, e.g. deoxyadenosine, deoxy cytidine, deoxy guanosine, deoxythymidine for DNA or their ribose counterparts for RNA, linked by phosphodiester linkages. They may also comprise non-natural nucleotide analogues, e.g. including modified bases, sugars, and / or internucleosidic linkages.

[0006] [3] Polynucleotides have a wide range of applications including DNA sequencing, gene editing, molecular probes and polymerase chain reaction. These polynucleotides are usually manufactured via phosphoramidite chemistry, which is performed by repeating cycles of passing reagents in a solvent over a column followed by cleavage and purification.

[0007] [4] However, phosphoramidite chemistry has several downsides such as the need to use a solvent, the duration of a cycle, the length of the product that is obtained at the end of a cycle and the purity of this product. In order to alleviate these downsides, enzymatic RNA synthesis (ERS) and enzymatic DNA synthesis (EDS) have been used as alternatives to phosphoramidite chemistry as they require no solvent and produce longer polynucleotides in a shorter period of time. ERS and EDS consist in elongating an oligonucleotide primer with a protected nucleotide, such elongation involving enzymes. The protected nucleotide is then deprotected to allow the insertion of the following part of the RNA or DNA sequence, and the process is repeated until a desired product is obtained.

[0008] [5] WO 2021 / 254934A1 discloses a device for EDS and ERS. In this device, and the associated process, an initiator is attached to a solid support. Reagents, including protected NTPs and a template-independent polymerase, are supplied to the initiator and are reacted to elongate the initiator through several cycles of elongation. The reactants are washed after each cycle of elongation such that the template-independent polymerase is lost during the wash step.

[0009] [6] There is therefore a need to provide a more efficient and cost-effective process and device for performing enzyme-driven synthesis of nucleic acids.

[0010] SUMMARY OF THE INVENTION

[0011] [7] The present invention provides methods and devices for template-free enzymatic synthesis of polynucleotides.

[0012] [8] In one aspect, the present invention is directed to a method of enzymatically synthesizing at least one polynucleotide, the method comprising:

[0013] (a) providing a reaction volume at least partly delimited by at least one filtering barrier;

[0014] (b) providing in the reaction volume at least one initiator nucleic acid and one template independent polymerase, wherein each initiator nucleic acid comprises a free 3 '-hydroxyl group;

[0015] (c) performing a cycle comprising the steps of: i) providing in the reaction volume 3’-0 reversibly blocked nucleoside triphosphates under suitable conditions for polymerase-mediated extension of the at least one initiator nucleic acid, or of an extension product thereof, by incorporation of a 3’-O-reversibly blocked nucleoside triphosphate, resulting in the production of a 3’-O-reversibly blocked extension product; ii) deprotecting the 3’-O-reversibly blocked extension product, thereby forming an extension product having a free 3 ’-hydroxyl group; and

[0016] (d) repeating the cycle of step (c) until synthesis of the at least one polynucleotide is complete, wherein, at step (c) the unincorporated 3’-O-reversibly blocked nucleoside triphosphates are removed from the reaction volume by diffusion through the filtering barrier, wherein the filtering barrier retains the at least one initiator nucleic acid and / or the template-independent polymerase in the reaction volume.

[0017] [9] In another aspect, the present invention is directed to a device to enzymatically synthesize at least one polynucleotide, the device comprising at least one reaction volume at least partly delimited by a filtering barrier.

[0018]

[0010] In another aspect, the present invention is directed to a device to enzymatically synthesize at least one polynucleotide, in particular according to the method of the invention, the device comprising a reaction volume at least partly delimited by a filtering barrier, one or more means for supplying controlled amounts of reagents for synthesis of polynucleotides to the reaction volume, wherein the device optionally comprises means for controlling temperature within the reaction volume, and one or more reservoirs for storing reagents for synthesis of polynucleotides.

[0019]

[0011] In another aspect, the present invention is directed to a kit to enzymatically synthesize at least one polynucleotide, in particular according to the method of the invention, wherein said kit comprises a device according to the invention and one or more reagents for synthesis of polynucleotides selected from one or more initiator nucleic acids, 3’-0 reversibly blocked nucleoside triphosphates, template-independent polymerase and, optionally, buffers and reagents for the synthesis of polynucleotides.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

[0012] Fig. 1 illustrates a schematic representation of steps of an embodiment of the method of the invention.

[0022]

[0013] Fig. 2 shows electrophoresis gels of different fractions resulting from DNA synthesis according to the method of the invention.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024]

[0014] In one aspect, the present invention is directed to a method of enzymatically synthesizing at least one polynucleotide, the method comprising:

[0025] (a) providing a reaction volume at least partly delimited by at least one filtering barrier;

[0026] (b) providing in the reaction volume at least one initiator nucleic acid and one template-independent polymerase, wherein each initiator nucleic acid comprises a free 3'-hydroxyl group;

[0027] (c) performing a cycle comprising the steps of: i) providing in the reaction volume 3’0-reversibly blocked nucleoside triphosphates under suitable conditions for polymerase-mediated extension of the at least one initiator nucleic acid, or of an extension product thereof, by incorporation of a 3 ’ -O-reversibly blocked nucleoside triphosphate, resulting in the production of a reversibly blocked extension product; ii) deprotecting the 3 ’ -O-reversibly blocked extension product, thereby forming an extension product having a free 3 ’-hydroxyl group; and (d) repeating the cycle of step (c) until synthesis of the at least one polynucleotide is complete, wherein, at step (c), the unincorporated 3’-O-reversibly blocked nucleoside triphosphates are removed from the reaction volume by diffusion through the filtering barrier, wherein the filtering barrier retains the at least one initiator nucleic acid and / or the template-independent polymerase in the reaction volume.

[0028]

[0015] As used herein, a “reaction volume” refers to any tridimensional space which can contain a solution, for instance a liquid solution. The reaction volume may be, for instance, a reaction vessel, a reaction compartment, a reaction chamber. In the context of the present invention, the reaction volume is configured such that the reaction steps of the method can be carried out in the reaction volume. The reaction volume may be connected with means for supplying reaction solutions and / or buffers. The reaction volume is at least partly delimited by at least one filtering barrier. The reaction volume may be fully delimited by at least one filtering barrier, i.e. entirely surrounded by the at least one barrier. Alternatively, the reaction volume may be partly delimited by at least one filtering barrier. The at least one filtering barrier is configured and positioned such that the 3’-O-reversibly blocked nucleoside triphosphates may be removed from the reaction volume by diffusion through the barrier. For instance, the at least one filtering barrier can fully or partially close an outlet of the reaction volume such as a tube adapted for removing the reagents. More than one filtering barriers may delimit the reaction volume, e.g. an inlet barrier and an outlet barrier. Various configurations for the barrier(s) may be readily implemented by the skilled person.

[0029]

[0016] In some embodiments, the 3’-O-reversibly blocked nucleoside triphosphates are supplied in the reaction volume by diffusion through at least one filtering barrier. Several filtering barriers may be used for different purposes, e.g. at least one filtering barrier for supplying the 3’-O- reversibly blocked nucleoside triphosphates in the reaction volume and at least one filtering barrier for removing the 3 ’ -O-reversibly blocked nucleoside triphosphates to the reaction volume.

[0030]

[0017] In some embodiments, further reagents are supplied in the reaction volume and / or removed from the reaction volume through the at least one filtering barrier. For instance, one or more of buffer molecules, salts and deblocking reagents can be supplied in the reaction volume and / or removed from the reaction volume.

[0031]

[0018] In some embodiments, the at least one initiator nucleic acid is in solution within the reaction volume. In other embodiments, the at least one initiator nucleic acid is attached to a solid support. In some embodiments, the polymerase is in solution within the reaction volume. In some embodiments, the at least one initiator nucleic acid and the polymerase are in solution within the reaction volume. A molecule is in solution when it is dispersed in a solution. In particular, the at least one initiator nucleic acid and / or the polymerase is not attached and / or immobilized to a solid support.

[0032]

[0019] In some embodiments, the initiator nucleic acid is attached to a protein. In particular, the initiator nucleic acid is attached to the polymerase. Attachment may be by covalent or non-covalent attachment. Methods of attachment are described, e.g. in Schneider et al., J. Am. Chem. Soc. 2023, 145, 38, 20874-20882, the entire content of which is hereby incorporated by reference.

[0033]

[0020] In some embodiments, the at least one initiator nucleic acid is retained within the reaction volume by the filtering barrier. In some embodiments, the polymerase is retained within the reaction volume by the filtering barrier. In some embodiments, the at least one initiator nucleic acid and the polymerase are both retained within the reaction volume by the filtering barrier.

[0034]

[0021] As used herein, an entity such as a biomolecule is said to be retained by the filtering barrier if at least 50%, preferably at least 60%, 70%, 80%, 90%, 95%, 99% or 99.9% of the entity does not diffuse through the filtering barrier, under elongation experimental conditions.

[0035]

[0022] In some embodiments, at least 50%, preferably 70%, still preferably 90%, more preferably at least 95%, even more preferably at least 99%, still more preferably at least 99.9%, most preferably at least 99.99% of the polymerase is retained within the reaction volume from one cycle of step (c) to the next cycle of step (c).

[0036]

[0023] In some embodiments, at least 50%, preferably 70%, still preferably 90%, more preferably at least 95%, even more preferably at least 99%, still more preferably at least 99.9%, most preferably at least 99.99% of the at least one initiator nucleic acid is retained within the reaction volume from one cycle of step (c) to the next cycle of step (c).

[0037]

[0024] In some embodiments, at least 90%, preferably at least 95%, more preferably at least 99%, still preferably at least 99.9%, most preferably at least 99.99% of the unincorporated 3’-O- reversibly blocked nucleoside triphosphates are removed from the reaction volume from one cycle of step (c) to the next cycle of step (c), by diffusion through the at least one filtering barrier. In particular, less than 0.1%, preferably less than 0.01%, more preferably less than 0.001% of the unincorporated 3’-O-reversibly blocked nucleoside triphosphates remain within the reaction volume from one cycle of step (c) to the next cycle of step (c).

[0025] Any combination of the indicated figures is encompassed by the invention. In particular, at least 90% of the polymerase and / or initiator nucleic acid is retained within the reaction volume and at least 90% of the unincorporated 3’-O-reversibly blocked nucleoside triphosphates are removed by diffusion through the at least one filtering barrier, from one cycle of step (c) to the next cycle of step (c). More particularly, at least 99% of the polymerase and / or initiator nucleic acid is retained within the reaction volume and at least 99% of the unincorporated 3’-O-reversibly blocked nucleoside triphosphates are removed by diffusion through the at least one filtering barrier, from one cycle of step (c) to the next cycle of step (c). Even more particularly, at least 99.9% of the polymerase and / or initiator nucleic acid is retained within the reaction volume and at least 99.9% of the unincorporated 3’-O-reversibly blocked nucleoside triphosphates are removed by diffusion through the at least one filtering barrier, from one cycle of step (c) to the next cycle of step (c).

[0038]

[0026] In some embodiments, the method does not comprise adding template-independent polymerase in the reaction volume during step (c). In some embodiments, the method does not comprise adding template-independent polymerase in the reaction volume for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive cycles of step (c), in particular at least 15, 20, 25, 30, 35, 40, 45 or 50 consecutive cycles of step (c). The method of the invention thus allows to reduce the amount of enzyme used during a polynucleotide synthesis, providing cost efficiency. Reduced enzyme supplying also reduces clogging that may be caused by the enzyme in the supplying tubes.

[0039]

[0027] In some embodiments, the filtering barrier is a physical, chemical or physico-chemical barrier.

[0040]

[0028] In some embodiments, the filtering barrier is a semi-permeable membrane. As used herein, a semipermeable membrane is a type of membrane, e.g. biological or synthetic or polymeric, that allows certain molecules or ions to pass through it by osmosis. Depending on the membrane and the solute, permeability may depend on solute size, solubility, properties, or chemistry.

[0041]

[0029] In some embodiments, the barrier is a membrane with pores.

[0042]

[0030] In some embodiments, the filtering barrier is selected from a microfiltration, ultrafiltration, nanofiltration and reverse osmosis membrane.

[0043]

[0031] In some embodiments, the filtering barrier is a molecular weight cut-off membrane.

[0044]

[0032] In some embodiments, the filtering barrier is a membrane which has a Molecular weight cutoff (MWCO) which is higher than the molecular weight of the 3’-O-blocked nucleoside triphosphate and lower than the molecular weight of the at least one initiator nucleic acid and / or the molecular weight of the template-independent polymerase, in particular lower than the molecular weight of the at least one initiator nucleic acid and lower than the molecular weight of the template-independent polymerase.

[0045]

[0033] In some embodiments, the filtering barrier is a membrane which has a Molecular weight cutoff (MWCO) of at least 1 kDa, in particular at least 1.5kDa, more particularly at least 2 kDa, still more particularly at least 3 kDa, even more particularly at least 5 kDa, most particularly at least 7 kDa. In some embodiments, the barrier is a membrane which has a Molecular weight cutoff (MWCO) of at least 0.75 kDa. In some embodiments, the barrier is a membrane which has a Molecular weight cutoff (MWCO) of at most 43 kDa, particularly at most 40 kDa, more particularly at most 30 kDa, still more particularly at most 10 kDa. Any combination of the indicated range ends is encompassed by the invention.

[0046]

[0034] In some embodiments, the barrier is a membrane which has a Molecular weight cutoff (MWCO) comprised between 1 and 43 kDa, particularly between 1.5 and 40 kDa, more particularly between 2 and 30 kDa, still more particularly between 3 and 10 kDa, even more particularly between 5 and 10 kDa, most particularly between 7 and 10 kDa. In some embodiments, the barrier is a membrane which has a Molecular weight cutoff (MWCO) comprised between 1 and 15 kDa, particularly between 1 and 10 kDa, more particularly between 1 and 7 kDa. In some embodiments, the barrier is a membrane which has a Molecular weight cutoff (MWCO) comprised between 2 and 15 kDa, particularly between 2 and 10 kDa, more particularly between 2 and 7 kDa. In some embodiments, the barrier is a membrane which has a Molecular weight cutoff (MWCO) comprised between 0.75 and 43 kDa. The cutoff of the membrane may be defined depending on the molecular weight of the initiator nucleic acid. A high cutoff can be selected for longer initiator nucleic acids. 5. In some embodiments, at least one filtering barrier is selected from a polymeric membrane, an inorganic membrane or a mixed polymeric-inorganic membrane. In some embodiments, the at least one filtering barrier is made of a material selected from cellulose acetate, regenerated cellulose, poly ethersulfone, polytetrafluoroethylene, polyvinylidene difluoride, polyethylene, polypropylene, polysulfone, polybenzimidazole, polyetheretherketone, polyacrylonitrile, polyamide, polyimide, polyetherimide, polyaniline, polypyrrole and mixtures thereof.

[0047] In some embodiments, the barrier is a compound that is not miscible with one or more of the initiators, the extension products and the template-independent polymerase. For example a lipidic barrier, e.g. based on oil, may serve as filtering barrier. Emulsions, such as water-in-oil emulsions can be used to configure a reaction volume delimited by a filtering barrier.

[0048]

[0035] In some embodiments, step ii) of step (c) is carried out while the dNTP is not present in the reaction volume anymore. In particular, after step i) of step (c), the unincorporated 3’-0 reversibly blocked nucleoside triphosphate may be removed from the reaction volume by diffusion through the barrier.

[0049]

[0036] In some embodiments, the polynucleotide is DNA.

[0050]

[0037] In some embodiments, the template-independent polymerase is a terminal deoxynucleotidyl transferase (TdT) or a variant thereof (see e.g. WO2017216472, W02019 / 135007, W02020 / 099451, WO2021 / 116270, WO2021 / 213903, WO2022 / 063835, WO2023083997, WO2020 / 239737 and WO2023 / 083999, the entire content of which is hereby incorporated by reference).

[0051]

[0038] In some embodiments, the polynucleotide is RNA.

[0052]

[0039] In some embodiments, the template-independent polymerase is a poly(A)polymerase (PAP) or poly(U)polymerase (PUP).

[0053]

[0040] In some embodiments, the method comprises washing the reaction volume after the elongation step i) and / or after the deprotecting step ii). In particular, the unincorporated 3’-O- reversibly blocked nucleoside triphosphates may be removed from the reaction volume by washing. In particular, the deblocking reagent may be removed from the reaction volume by washing. In particular, the method may comprise supplying a washing buffer into the reaction volume.

[0054]

[0041] In some embodiments, the step of deprotecting the 3’-O-reversibly blocked extension product comprises providing a deblocking reagent which reacts with the 3 ’ -O-protecting group of the 3’-O-reversibly blocked extension product. In particular, the deblocking reagent is supplied in and / or removed from the reaction volume by diffusing through the filtering barrier. More particularly, the deblocking reagent may be removed from the reaction volume after step ii) of step c).

[0055]

[0042] In some embodiments, the deblocking agent is a phosphonate compound, in particular a phosphonate compound having the formula R-P(=0)(0M)0M (“Formula I”) 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 H or 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(Ri)3 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; and R 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.

[0056]

[0043] Phosphonate compounds are suitable to deprotect 3’-O-amino protected groups. Examples of phosphonate compounds, in particular of carbonylphosphonate compounds, are described in International patent application PCT / EP2024 / 062042, the content of which is incorporated by reference in its entirety.

[0057]

[0044] Preferably, 3’0-amino blocking group is deblocked by application of a phosphonate compound, e.g. a carbonylphosphonate compound. Most preferably, 3’0-amino blocking group is deblocked by application of a carbonylbisphosphonate compound.

[0058]

[0045] In another embodiment, the deblocking reagent is a phosphine, preferably Tris(3- hydroxypropyl)phosphine THPP or tris(2-carboxyethyl)phosphine TCEP.

[0059]

[0046] Alternatively, a deblocking reagent may be an enzymatic deblocking reagent, 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 the deblocking reagent depends on the type of 3 ’-nucleotide blocking group used, whether one or multiple blocking groups are being used, whether initiator nucleic acids are attached to living cells or organisms, that may necessitate mild treatment, or to solid supports, and the like.

[0060]

[0047] In some embodiments, the duration of the deprotection step is comprised between 30 minutes and 120 minutes, preferably between 40 minutes and 100 minutes.

[0061]

[0048] In some embodiments, the initiator comprises at least one cleavable group. In some embodiments, the method comprises a further step of cleaving said at least one cleavable group, thereby releasing the at least one polynucleotide from the initiator.

[0062]

[0049] In particular, the at least one cleavable group may be an enzymatically cleavable group. The cleavable group may be selected from a photocleavable group, a chemically cleavable group, an enzymatically cleavable group, and their combinations. Enzymatically cleavable groups may be selected from the group consisting in a deoxyuridine (dU), a deoxyinosine (di), an inosine, an uracil, a nitroindole, a hypoxanthine, a thymidine glycol, a 5-hydroxyuracil, 5,6-dihydrouracil, a 5-hydroxycytosine and their combinations.

[0050] In some embodiments, the at least one initiator comprises at least one capture tag. In some embodiments, the method comprises a further step of capturing the at least one polynucleotide through binding of the capture tag. Said step of capturing may occur after or before a step of cleaving.

[0063]

[0051] In some embodiments, the capture tag is an amino acid tag, for instance selected from a poly-amino acid tag, a poly-His tag, a 6His-tag or the like; a chemical compound such as a polyethylene glycol; a protein involved in a protein-protein binding pair, such as biotin-avidin; an affinity coupling tag; a capture probe; a hydrophobic group; or any combination of these.

[0064]

[0052] In some embodiments, the reaction volume is comprised between 0,5 mL and 70 mL.

[0065]

[0053] In some embodiments, the quantity of initiators in the reaction volume is comprised between 2 nmol and 100 nmol.

[0066]

[0054] In some embodiments, the method comprises a step of centrifugation at step (c), preferably after step i) and / or ii) of step (c). The speed of centrifugation may be at least 5000 xg, preferably 10000 xg, more preferably 12000xg. The duration of the centrifugation may be of at least 5 min, preferably at least 10 min, more preferably at least 15 min. Centrifugation at an adapted speed may separate the unincorporated 3’-O-reversibly blocked nucleoside triphosphates from the initiator nucleic acid and / or template- independent polymerase.

[0067]

[0055] In some embodiments, the method is carried out with a step of centrifugation at step (c), preferably after step i) and / or ii) of step (c), wherein centrifugation is carried out with a filtering barrier, wherein centrifugation combined with filtration separates the unincorporated 3’-O- reversibly blocked nucleoside triphosphates from the initiator nucleic acid and / or templateindependent polymerase.

[0068]

[0056] The term ‘polynucleotide’ as used herein refers to a polymer of nucleotides. Suitably to a polymer of A, T, U, G or C nucleotides, or optionally modified or synthetic nucleotides or nucleotide analogues comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. The terms "polynucleotide(s)", "nucleic acid sequence(s)", "nucleotide sequence(s)", "nucleic acid(s)", "nucleic acid molecule" are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.

[0069]

[0057] In some embodiments, a single template-independent polymerase variant may be employed for coupling all nucleotides in the synthesis of a polynucleotide. Alternatively, multiple different template-independent polymerase variants may be employed for coupling different nucleotides in the synthesis of a polynucleotide. In some embodiments therefore the methods of synthesising a polynucleotide may comprise contacting with more than one template-independent polymerase variant enzyme of the invention.

[0070]

[0058] In some aspects of the invention, 3’-O-protected nucleotide triphosphates are used, in particular 3’-O-reversibly blocked nucleoside triphosphates. Guidance in selecting 3’-O-protecting groups and corresponding deprotecting conditions for the above method may be found in the following references: U.S. patent 5808045; U.S. patent 8808988; International patent publication WO91 / 06678, for example.

[0071]

[0059] In some embodiments, it is desirable to employ two or more different protecting groups that may be removed using orthogonal deprotection conditions. The following exemplary pairs of protecting groups may be used in parallel synthesis embodiments in which two or more polynucleotide sequences are synthesised in the same reaction mixture. It is understood that other protecting group pairs, or groups containing more than two protecting groups, may be available for use in the invention.

[0072]

[0060] Table 2: Protecting Group Pairs

[0073]

[0061] In some embodiments, if the polynucleotide to be synthesised is RNA, the protected nucleotide is an rNTP (ribonucleoside triphosphate). In some embodiments, the elongation step may comprise between 125-600 pM protected rNTP. Preferentially protected rNTPs may be 3’-O- blocked rNTPs, which may be selected from protected A, C, G and U ribonucleosides. In some embodiments, the protection group is selected from a 3’-O-propargyl, a 3’-O-azidomethyl, 3’-O- amino, 3’-O-allyl, 3’-O-methyl, 3 ’-O-(2 -nitrobenzyl), 3’-O-tert-butoxy ethoxy and a 3’-O-(2- cyanoethyl) group. The 3’-O-blocked rNTPs employed in the invention may be purchased from commercial vendors (e.g. Jena Bioscience, MyChemLabs, or the like) or synthesized using published techniques, e.g. U.S. patent 7057026; International patent publications W02004 / 005667, WO91 / 06678; Canard et al, Gene (cited above); 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; Zavgorodny et al, Tetrahedron Letters, 32(51): 7593-7596 (1991).

[0074]

[0062] In some embodiments, if the polynucleotide to be synthesised is DNA, the protected nucleotide is a dNTP (deoxyribonucleoside triphosphate). In some embodiments, the elongation step may comprise between 125-600 pM protected dNTP. Preferentially protected dNTPs may be 3’-O-blocked dNTPs, which may be selected from protected A, C, G and U deoribonucleosides. In some embodiments, the protection group is selected from a 3’-O-propargyl, a 3 ’ -O-azidomethyl, 3’- 0-NH2, 3’-O-allyl, 3’-O-methyl, 3 ’-O-(2 -nitrobenzyl), 3’-O-tert-butoxy ethoxy and a 3’-O-(2- cyanoethyl) group. Preferentially, the blocking group is a 3’-O-amino protecting group. In some embodiments, the deblocking reagent to deprotect a 3’-O-amino protecting group is a phosphonate compound as described in the present specification.

[0075]

[0063] Phosphonate compounds are able to deprotect the polynucleotide under mild conditions and are advantageous deprotection reagents to preserve the integrity of filtering barriers, e.g. ultrafiltration membranes such as polymeric membranes.

[0076]

[0064] In some embodiments, the nucleic acid initiator may further comprise one or more fluorescent groups or tags.

[0077] Template-Free Enzymatic Synthesis

[0078]

[0065] 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 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: 0.1039 / c6ob01371f (2016); Schmitz et al, Organic Lett. , 1(11): 1729-1731 (1999).

[0079]

[0066] Initiator nucleic acids (100) are provided which have free 3 ’-hydroxyl groups (103). A template-independent polymerase (104), such as a TdT or a variant thereof (e.g. WO / 2017 / 216472, W02019 / 135007, W02020 / 099451, WO2021 / 116270, WO2021 / 213903, WO2022 / 063835, WO2023083997, WO2020 / 239737 and WO2023 / 083999) is also provided. Those components are retained by a filtering barrier (102) through which, by contrast, smaller molecules like 3’-O- protected-NTP may diffuse.

[0080]

[0067] In a first step, to the initiator nucleic acids (100) (or elongated initiator nucleic acids in subsequent cycles) and template-independent polymerase (104) are added a 3’-O-reversibly blocked-NTP under conditions effective for the enzymatic incorporation of the 3’-O-reversibly blocked-NTP onto the 3’ end of the initiator nucleic acids (100) (or elongated initiator nucleic acids which are extension products of the initiator nucleic acids). This reaction produces elongated initiator nucleic acids whose 3’-hydroxyls are protected (106).

[0081]

[0068] Subsequently, if the elongated initiator nucleic acid does not contain a completed sequence, then the 3’-O-protection groups are removed to expose free 3 ’-hydroxyls (103) and the elongated initiator nucleic acids are subjected to another cycle (108) of nucleotide addition and deprotection. In case the elongated initiator nucleic acid contains a completed sequence, the 3’-O-protection group may be deprotected. The desired sequence may be cleaved from the initiator nucleic acid (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 initiator nucleic acid. An exemplary cleavable nucleotide may be an uracil nucleotide which is cleaved by uracil DNA glycosylase.

[0082]

[0069] 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 a moiety that 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 extension product (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 extension product (or “extension intermediate”) by an enzymatic coupling reaction.

[0083]

[0070] 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 double-stranded. 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.

[0084]

[0071] 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.

[0085]

[0072] Returning to Fig. 1, in some embodiments, an ordered sequence of nucleotides are coupled to an initiator nucleic acid using a template-independent polymerase, such as TdT, in the presence of 3’-O-protected dNTPs in each synthesis step. In some embodiments, the method of synthesizing a polynucleotide comprises the steps of (a) providing at least one initiator nucleic acid (100) having a free 3’-hydroxyl group (103); (b) reacting under extension conditions the at least one initiator nucleic acid or an extension intermediate having a free 3 ’-hydroxyl (103) with a templateindependent polymerase (104) in the presence of a 3’-O-reversibly blocked nucleoside triphosphate to produce a 3’-O-reversibly blocked extension intermediate (106); (c) deprotecting the extension intermediate to produce an extension intermediate with a free 3 ’-hydroxyl (108); and (d) repeating steps (b) and (c) until the polynucleotide is synthesized. (The terms “extension intermediate” and “elongation fragment” may be used interchangeably). In some embodiments, an initiator is provided as a polynucleotide attached to a solid support, e.g. by its 5’ end. The above method may also include washing steps after each reaction, or extension, step, as well as after each deprotecting 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 last from a few seconds, e.g. 30 sec, to several minutes, e.g. 30 min.

[0086]

[0073] As used herein, an unincorporated nucleoside triphosphate such as an unincorporated 3’- O-reversibly blocked nucleoside triphosphate is a nucleoside triphosphate which has not been added to the 3 ’-OH end of the initiator or extension fragment thereof, by template-independent polymerase reaction.

[0087]

[0074] When the sequence of an enzymatically synthesized polynucleotide 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 preventing the 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 initiator nucleic acid.

[0088]

[0075] 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 templateindependent 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.

[0089]

[0076] 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.

[0090]

[0077] 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.

[0091]

[0078] When base-protected dNTPs are employed, the enzymatic synthesis 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.

[0092]

[0079] The above methods 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 step, 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 deblocked 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 strands that fail to be deblocked inert to any subsequent coupling or deprotection. 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.

[0093]

[0080] 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: from 2.0 pM to 50 pM purified TdT, preferably about 10 pM purified TdT; from 125 pM to 600 pM 3’-O-blocked dNTP (e.g. 3’-O-NH2-blocked dNTP); from 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. C0CI2 or MnC U), where the elongation reaction may be carried out in a 50 pL reaction volume, at a temperature within the range of from room temperature to about 45 °C, for a duration of, e.g., 3 minutes. In embodiments, in which the 3’-O-blocked NTPs are 3’-O-NH2-blocked dNTPs, reaction conditions for a deprotecting 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 deprotecting reaction may be carried out in a 50 pL volume, at a temperature within the range of from room temperature to 45°C for a duration of about 30 seconds to several minutes.

[0094]

[0081] In some embodiments, e.g. employing a phosphonate compound such as a carbonylphosphonate and its salts as deblocking reagents, an effective amount is provided by a concentration in the range of from 0.1 to 500 mM, or in other embodiments in the range of from 0.1 to 200 mM, or in other embodiments in the range of from 0.1 to 100 mM. Stated otherwise, the molar ratio between the polynucleotides and the deblocking reagent typically ranges from 1 : 1 to 1 : 1000 and preferably from 1:1 to 1 :100.

[0095]

[0082] The deblocking reagent is typically provided in an aqueous solution buffered at a pH of from 4 to 8, preferably from 5 to 7 (i.e. buffer), wherein the buffering agent may be selected from citrates, phosphates such as sodium phosphate, acetates such as sodium acetate or bicarbonates.

[0096]

[0083] According to a preferred embodiment of this invention, the deprotection buffer may further include at least one inorganic salt of a divalent metal such as magnesium, calcium, zinc or copper, preferably magnesium sulfate, which has been found to increase the deprotection yield, especially at higher pH. This compound may be comprised within the buffer in an amount ranging from 1 to 100 equivalents, preferably from 1 to 10 equivalents, with respect to the deblocking reagent.

[0097]

[0084] In addition to water, the deprotection buffer may further include at least one organic solvent which is miscible with water, especially a polar protic solvent, such as methanol or ethanol (up to about 50% v / v), or a polar aprotic solvent, such as tetrahydrofuran or dioxane (up to about 40% v / v). Alternatively, or in addition to these organic solvents, the buffer may also include one or more denaturants, such as formamide, urea, dimethylformamide or dimethylsulfoxide (up to about 25%).

[0098]

[0085] Washes may be performed with the cacodylate buffer without the components of the coupling reaction (e.g. enzyme, monomer, divalent cations).

[0099]

[0086] Depending on particular applications, the steps of deprotecting 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- protecting groups and corresponding deprotecting 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 WO91 / 06678; and references cited below. In some embodiments, the cleaving agent (also sometimes referred to as a deblocking reagent or agent or deprotecting reagent or agent) is a chemical cleaving agent, such as, for example, dithiothreitol (DTT). It will be understood by the person skilled in the art that the selection of deblocking reagent depends on the type of 3 ’-nucleotide blocking group used, whether one or multiple blocking groups are being used, , and the like, that necessitate mild treatment. For example, the selection of the deblocking reagent will also depend on the initiator nucleic acid and / or polymerase being retained by the filtering barrier for a next cycle of extension, thus needing to preserve the initiator nucleic acid and / or the polymerase, as well as the filtering membrane. A phosphine, such as tris(2-carboxyethyl)phosphine (TCEP) can be used to cleave a 3’O-azidomethyl groups, palladium complexes can be used to cleave a 3’O-allyl groups. In particular embodiments, the cleaving reaction involves TCEP, a palladium complex. Phosphonate compounds, such as carbonylphosphonates can be used to cleave a 3’-O-amino group.

[0100]

[0087] 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 enzymatically synthesizing at least one polynucleotide, the method comprising:

[0101] (a) providing a reaction volume at least partly delimited by at least one filtering barrier;

[0102] (b) providing in the reaction volume at least one initiator nucleic acid and one template-independent polymerase, wherein each initiator nucleic acid is a 3’-O-reversibly blocked initiator nucleic acid;

[0103] (c) performing a cycle comprising the steps of: i) deprotecting the 3’-O-reversibly blocked initiator nucleic acid or an extension product thereof, thereby forming an initiator nucleic acid or extension product having a free 3’- hydroxyl group; and ii) providing in the reaction volume 3’O-reversibly blocked nucleoside triphosphates under suitable conditions for polymerase-mediated extension of the at least one initiator nucleic acid, or extension product thereof, by incorporation of a 3’-O-reversibly blocked nucleoside triphosphate, resulting in the production of a reversibly blocked extension product;

[0104] (d) repeating the cycle of step (c) until synthesis of the at least one polynucleotide is complete, wherein, at step (c), the unincorporated 3’-O-reversibly blocked nucleoside triphosphates are removed from the reaction volume by diffusion through the filtering barrier, wherein the filtering barrier retains the at least one initiator nucleic acid and / or the template-independent polymerase in the reaction volume.

[0105] Polynucleotide Products

[0106]

[0088] Some aspects of the invention relate to a polynucleotide produced from a method employing the template-independent polymerase variants, and further to compositions comprising said polynucleotide products.

[0107]

[0089] 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). In some embodiments the polynucleotide may be selected from ssRNA, ssDNA, dsRNA, and dsDNA.

[0108]

[0090] 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.

[0109]

[0091] 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 in length, up to 60 nucleotides in length, up to 50 nucleotides in length, or up to 40 nucleotides in length.

[0110]

[0092] In one or more embodiments, the polynucleotide product may comprise a high number of C and / or G nucleotides (high GC content). In these embodiments, the polynucleotide may be classified as being a ‘difficult’ sequence for the template-free polymerase variant to synthesise. 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%, or at least 75% of C and / or G nucleotides. In some embodiments, the polynucleotide may comprise a majority of C and / or G nucleotides, for example over 50% of C and / or G nucleotides.

[0111]

[0093] The term “Molecular weight cut-off’ (MWCO) relates to a method of characterization used in filtration to describe pore size distribution and retention capabilities of membranes. It is defined as the lowest molecular weight (in Daltons) at which greater than 90% of a solute with a known molecular weight is retained by the membrane.

[0112]

[0094] The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art. Such conventional techniques may include, but are not limited to, preparation and use of synthetic peptides, synthetic polynucleotides, monoclonal antibodies, nucleic acid cloning, amplification, sequencing and analysis, and related techniques. Protocols for such conventional techniques can be found in product literature from manufacturers and in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV); PCR Primer: A Laboratory Manual; and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Lutz and Bornscheuer, Editors, Protein Engineering Handbook (Wiley-VCH, 2009); Hermanson, Bioconjugate Techniques, Second Edition (Academic Press, 2008); and like references.

[0113] Device for synthesizing biomolecules

[0114]

[0095] Hereafter is provided in a non-limiting way, a specific device according to the invention, adapted to implement specific embodiments. In particular, the present disclosure provides a device for synthesizing biomolecules, equipped with a filtration device comprising a first chamber connected to a first loop, a second chamber connected to a second loop, the chambers being arranged coaxially in respect with each other, and at least one filtering barrier, such as a semi- permeable membrane separating radially the first chamber and the second chamber. The first chamber and / or the second chamber define a reaction volume. The first loop comprises a first pump connecting the first chamber to a single reservoir and the second loop comprises a second pump connecting the second chamber to a plurality of reservoirs, the first loop, the second loop and the at least one filtering barrier being configured to allow matter exchange through the at least one filtering barrier in a first direction and in a second direction alternatively.

[0115]

[0096] Filtration is a method which can be used to separate small molecules from larger molecules relying on parameters such as concentration, pressure, temperature and / or the surface available for the reaction. Matter can thus be exchanged through a membrane, here the at least one filtering barrier, with suspended solids and solutes of high molecular weight being retained on one side of the membrane called the retentate, and water and solutes of low molecular weight passing through to another side of the membrane called the permeate. Filtration has traditionally been used to separate and purify biomolecules such as RNA and DNA, but it could also be used to manufacture them on a large scale as is the case according to the present invention.

[0116]

[0097] The device according to the invention is thus capable of synthesizing biomolecules such as RNA and DNA polynucleotides using this filtration approach. To this end, a flow of matter comprising reactants is exchanged through the at least one filtering barrier between the first chamber and the second chamber, this flow following either a first direction or a second direction. First direction and second direction are opposite directions; for instance, matter circulating according to the first direction could mean that it diffuses from the first chamber to the second chamber, and matter circulating according to the second direction could mean that it diffuses from the second chamber to the first chamber.

[0117]

[0098] The first chamber and second chamber can be coaxial and are separated by the at least one filtering barrier, meaning that one of the chambers is surrounded by the at least one filtering barrier, which is itself surrounded by the other chamber.

[0118]

[0099] The first chamber is connected to a first loop and the second chamber is connected to a second loop. Each chamber is also connected to a pump, with the first chamber being connected to a first pump and the second chamber being connected to a second pump. It is understood by “connected” that each of the loops comprises pipes running between each chamber and its respective pump, while other components of the device might be inserted on the loops between the chambers, the reservoirs and their respective pumps.

[0119]

[0100] The pumps are used to circulate the contents of the reservoirs in the loops, in order to bring them to the first or the second chamber so that at least part of the contents of the single reservoir can be exchanged with at least part of the contents of the plurality of reservoirs through the at least one filtering barrier. 1

[0120]

[0101] The polynucleotide synthesis relies on an elongation process according to which reactants diffuse through the at least one filtering barrier to elongate biomolecules . As such, different reactants may go from one chamber to the other and reciprocally. For instance, nucleotides initially provided in the first loop may cross the at least one filtering barrier to reach the biomolecules contained in the second chamber. Once the elongation has taken place, supernumerary nucleotides which have not participated in this elongation process must cross the at least one filtering barrier once again in the opposite direction, such process necessitating other reactants. Furthermore, as the nucleotides used for elongation are protected as will be further described hereinafter, their deprotection involves still other reactants. This plurality of reactants and the need to have them interact with the contents of the first loop in a predetermined order justifies the need for a plurality of reservoirs in the second loop. As an embodiment of the invention, the first loop and the second loop are structurally distinct from each other.

[0121]

[0102] “Structurally distinct” means that each loop has dedicated pipes, with the matter circulating in the first loop using different pipes from the matter circulating in the second loop.

[0122]

[0103] According to an embodiment of the invention, the second loop comprises at least one regulation means.

[0123]

[0104] According to another embodiment of the invention, the at least one regulation means is a valve connected to each of the plurality of reservoirs.

[0124]

[0105] The at least one regulation means is used to control the flow in the second loop, for example through computer control. It can be configured to regulate the flow of the contents of each reservoir of the plurality of reservoirs separately, so that these contents circulate in the second loop one at a time and can be exchanged through the at least one filtering barrier in a predetermined order.

[0125]

[0106] In some embodiments, the single reservoir of the first loop contains at least one initiator nucleic acid.

[0126]

[0107] Such initiator nucleic acid is a polynucleotide, e.g. from 3 to 100 nucleotides, with a free 3 ’-hydroxyl at its end. It can be as an RNA initiator nucleic acid or a DNA initiator nucleic acid. It is to be elongated to obtain the desired synthesized polynucleotide, such elongation involving nucleotides contained in the second loop.

[0127]

[0108] According to an embodiment, the single reservoir of the first loop contains an elongation enzyme.

[0109] The synthesis of biomolecules taking place in the device requires an elongation of the above-mentioned initiator nucleic acid. To this end, the single reservoir contains a templateindependent polymerase whose role is to elongate the initiator nucleic acid. Such templateindependent polymerase is preferably a TdT or variants thereof.

[0128] [HO] In some embodiments, each reservoir of the plurality of reservoirs contains a reaction buffer.

[0129]

[0111] Each reservoir of the plurality of reservoirs of the second loop may contain the same reaction buffer, which can be tris hydrochloride also known as TrisHCl.

[0130]

[0112] According to another embodiment, the plurality of reservoirs of the second loop comprises at least one elongation reservoir containing nucleotides, a wash reservoir and a deprotection reservoir.

[0131]

[0113] The at least one elongation reservoir contains modified nucleotides, which are nucleotides associated with 3 ’-OH protecting groups so that they are 3’-O-blocked nucleotides. Protection of the nucleotides ensures that the initiator fragment can be elongated with one nucleotide at a time, permitting the synthesis of a polynucleotide having a desired sequence

[0132]

[0114] According to another embodiment of the invention, there are multiple elongation reservoirs, with each elongation reservoir containing a different 3’-0 reversibly blocked nucleoside triphosphate. In some embodiments, one elongation reservoir contains 3’-0 reversibly blocked adenosine triphosphate, one elongation reservoir contains 3’-0 reversibly blocked cytidine triphosphate, one elongation reservoir contains 3 ’-0 reversibly blocked guanosine triphosphate and one elongation reservoir contains 3’-0 reversibly blocked uridine triphosphate and / or one elongation reservoir contains 3’-0 reversibly blocked thymidine triphosphate.

[0133]

[0115] 3’-0 reversibly blocked uridine triphosphate would be used to elongate an RNA initiator fragment, while 3’-0 reversibly blocked thymidine triphosphate would be used in DNA synthesis.

[0134]

[0116] According to another embodiment of the invention, the elongation reservoir contains a metal cofactor.

[0135]

[0117] For example, such metal co-factor could be manganese chloride or cobalt chloride. Its role is to assist the elongating enzyme in adding the modified nucleotide to the initiator fragments.

[0136]

[0118] According to an embodiment of the invention, the wash reservoir contains lithium chloride and a non-ionic surfactant.

[0119] The deprotection reservoir typically contains a deprotection buffer. According to an embodiment of the invention, the deprotection reservoir contains a deblocking reagent suitable for deprotection of a protection group selected from 3’-O-propargyl, a 3’-O-azidomethyl, 3’-O- NH2, 3’-O-allyl, 3’-O-methyl, 3 ’ -O-(2 -nitrobenzyl), 3 ’ -O-tert-butoxy ethoxy and a 3’-O-(2- cyanoethyl) group, preferably a 3’-O-amino group. In some embodiments, the deprotection reservoir contains reducing reagent. In some embodiments, the deprotection reservoir contains nitrite, in particular sodium nitrite. In some embodiments, the deprotection contains a phosphonate compound of Formula I, as described herein, in particular a carbonylbisphosphonate compound. The deprotection reservoir may also contain a buffering agent, in particular selected from citrates, phosphates such as sodium phosphate, acetates such as sodium acetate or bicarbonates. The deprotection reservoir may also contain salts necessary for the deprotection reaction.

[0137]

[0120] According to an embodiment of the invention, the matter exchange through the at least one filtering barrier depends on the concentration of nucleotides in the first loop and in the second loop and / or the concentration of reducing agent in the first loop and in the second loop.

[0138]

[0121] In some embodiments, the first pump and the second pump are peristaltic pumps.

[0139]

[0122] As for the at least one regulation means, these peristaltic pumps can be under computer control.

[0140]

[0123] According to another embodiment of the invention, the first pump and the second pump are configured to be activated independently from one another.

[0141]

[0124] This means that the first pump can be controlled independently from the second pump; for instance, the first pump could be activated to create a continuous flow of matter from the single reservoir to the filtration device and back, while the second pump could be switched on and off to alternate between each one of the plurality of reservoirs in order to send their contents to the filtration device in a predetermined order.

[0142]

[0125] According to another embodiment of the invention, the device comprises at least one waste management unit.

[0143]

[0126] Such waste management unit could be used to discard the remaining reactants or to recycle them.

[0144]

[0127] According to an embodiment of the invention, the at least one waste management unit is located at an outlet of the second chamber.

[0128] According to an alternative, there are multiple waste management units, each one being directly connected to one of the reservoirs of the plurality of reservoirs.

[0145]

[0129] When there are multiple waste management units each located at an outlet of each one of the plurality of reservoirs, the outlet of the second chamber is connected to each one of these multiple waste management units through their respective reservoirs.

[0146]

[0130] According to another embodiment of the invention, the filtration device comprises a heating system.

[0147]

[0131] This heating system is located in the device in the vicinity of the chambers; as such, it may be arranged around both the first chamber and the second chamber.

[0148]

[0132] According to another embodiment of the invention, the device comprises a purification loop.

[0149]

[0133] Such purification loop can be used to separate the biomolecules from other reactants, and especially from the polymerase.

[0150]

[0134] According to an embodiment of the invention, the device comprises a desalting loop.

[0151]

[0135] According to an embodiment of the invention, the at least one filtering barrier is selected from a polymeric membrane, an inorganic membrane or a mixed polymeric-inorganic membrane.

[0152]

[0136] According to an embodiment of the invention, the at least one filtering barrier is made of a material selected from cellulose acetate, regenerated cellulose, polyethersulfone, polytetrafluoroethylene, polyvinylidene difluoride, polyethylene, polypropylene, polysulfone, polybenzimidazole, polyetheretherketone, polyacrylonitrile, polyamide, polyimide, poly etherimide, polyaniline, polypyrrole and mixtures thereof.

[0153]

[0137] The device for synthesizing biomolecules may include a solid support such as nanoparticles or beads, which allows the immobilization of the initiator nucleic acid during and after the elongation process.

[0154]

[0138] In some embodiments, the pores of the at least one filtering barrier are of a size small enough not to let the initiator nucleic acid diffuse through the filtering barrier.

[0155]

[0139] In some embodiments, the pores of the at least one filtering barrier are of a size small enough not to let the polymerase diffuse through the filtering barrier.

[0156]

[0140] In embodiments where the polymerase is contained in the single reservoir, it is thereby confined in the first loop and it cannot pass through the at least one filtering barrier from the first chamber to the second chamber. Alternatively, the polymerase may be confined in the second loop in embodiments where it circulates in this second loop.

[0157]

[0141] According to another embodiment of the invention, the pores have a Molecular weight cutoff (MWCO) of at least 0.75 kDa, in particular 1 kDa, in particular at least 1.5kDa, more particularly at least 2 kDa, still more particularly at least 3 kDa, even more particularly at least 5 kDa, most particularly at least 7 kDa. In some embodiments, the barrier is a membrane which has a Molecular weight cutoff (MWCO) of at most 55 kDa, particularly at most 45 kDa, more particularly at most 30 kDa, still more particularly at most 10 kDa. Any combination of the indicated range ends is encompassed by the invention.

[0158]

[0142] In some embodiments, the at least one filtering barrier is heat resistant at the temperatures at which the polynucleotide synthesis occurs.

[0159]

[0143] It is understood by “heat resistant” that the at least one filtering barrier can withstand temperatures at which the polynucleotides are synthesized. Such temperatures can range from 30 °C to 70°C .

[0160] The present invention is also directed to a kit to enzymatically synthesize at least one polynucleotide, wherein said kit comprises a device as provided in the present invention and one or more reagents for synthesis of polynucleotides.

[0161] The reagents may be selected from one or more of initiator nucleic acids, 3’-0 reversibly blocked nucleoside triphosphates, a template-independent polymerase, in particular TdT or a variant thereof. The reagents may also comprise buffers for the synthesis of polynucleotides as well as any reagent for the synthesis of polynucleotides. The buffers are preferably selected from an elongation buffer and a deprotecting buffer. In preferred embodiments, the deprotecting buffer comprises a deblocking reagent, as described herein. The present specification provides further examples of such buffers and reagents. Other examples will be apparent to the skilled person, for instance, from the references cited herein.

[0162] EXAMPLES

[0163]

[0144] The following examples are put forth 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 and 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.

[0164] Example 1: 2-cycles DNA synthesis using Amicon filter tubes

[0165]

[0145] This DNA synthesis was realized using an Amicon centrifugation device (Millipore, 0.5 mL, regenerated cellulose membrane, 3 kDa cut off) from Merck.

[0166] Elongation 1:

[0167]

[0146] 20 pL of TdT (from 250 pM stock, 10 pM final) and 5 pL of fluorescent initiator DNA (iDNA) 3 Omer (100 pM stock, 1 pM final) were added in the Amicon. In addition to this first experiment at a scale of 500 pmoles of iDNA, two further experiments were conducted at the scales of 50 and 2000 pmoles by using 0.5 pL or 20 pL of the fluorescent iDNA stock instead of 5 pL, respectively. The volume was subsequently completed to 500 pL with elongation buffer (500 mM sodium Cacodylate (“Caco”) pH 7.4, 4 mM C0CI2, 0.01% Tween 20, 15 mM Benzylhydroxylamine, 500 pM ONH2-dATP). The mix was incubated for 5min at room temperature, 800 rpm on thermomixer. 10 pL of reaction mix were stored for analysis. The reaction tube was centrifuged for about 20 min, 15000 xg to reduce the mix volume to ~50 pL.

[0168] Wash 1:

[0169]

[0147] 450 pL of wash buffer (200 mM Caco pH 7,4, 500 mM LiCl, 0.01% Tween 20, 15 mM Benzylhydroxylamine, 8 mM EDTA) were added to the mix and incubated 2 min at room temperature, 800 rpm on thermomixer. The reaction tube was centrifuged for about 20 min, 15000 xg to reduce the mix volume to about 50 pL.

[0170] Deprotection 1:

[0171]

[0148] 450 pL of deprotection buffer (20 mM Carbonyl diphosphonate, 1 M LiCl) were added to the reaction mix and incubated 5min at room temperature, 800 rpm on thermomixer. The reaction tube was centrifuged for about 20 min, 15000 xg to reduce the mix volume to ~50 pL. 10 pL of the centrifugation flow through were stored for analysis.

[0172] Wash 2:

[0173] 450 pL of wash buffer were added to the mix and incubated 2min at rt, 800 rpm on thermomixer. The reaction tube was centrifuged for about 20 min, 15000 xg to reduce the mix volume to about 50 pL.

[0174] Elongation 2:

[0149] 450 jj.L of elongation buffer were added to the Amicon tube containing the TdT, iDNA mix. The mix was incubated for 5min, 800 rpm on thermomixer. 10 pL of reaction mix were stored for analysis.

[0175] Results:

[0176]

[0150] The top panel of Figure 2 shows an agarose gel and the below panel shows an SDS page gel done in denaturing conditions with the different samples stored at the different stages of the experiments: EB1 reaction mix, DB flow through and EB2 reaction mix. The amount of initiator nucleic acid is varied from 50 to 2000 pmol as reflected by the scale line in the Figure.

[0177]

[0151] As reflected by the agarose gel, elongation was carried out quantitatively for 2 cycles of elongation, without fresh addition of polymerase or initiator nucleic acid during the elongation cycles, reflecting that most initiator nucleic acid and polymerase were successfully retained in the reaction mix by the filtering membrane. After 2 cycles of elongation, all polynucleotides were elongated by 2 nucleotides only. The absence of uncontrolled elongation by more than 2 nucleotides reflects that the ONEE-dATP which were unincorporated after the first elongation cycle, were entirely removed through the filtering membrane. On the bottom panel the SDS PAGE gel shows the presence of the polymerase in the reaction mix during the first elongation step (EB1) and the second elongation step (EB2) although no fresh polymerase has been added to the reaction mix between the two elongations. No polymerase has leaked through the membrane during the experiment.

[0178]

[0152] This experiment shows that successive cycles of TdT-driven elongation can be performed quantitatively by using initiator DNA and polymerase in solution, without addition of fresh polymerase at each cycle. The 3’-0 reversibly blocked nucleotides were successfully removed from the reaction volume by filtration. Further improvements are expected by tuning the cut-off value of the filter according to the molecular weight of the initiator nucleic acid and the polymerase and the scale of the synthesis being performed.

[0179]

[0153] REFERENCES

[0180]

[0154] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS1. A method of enzymatically synthesizing at least one polynucleotide, the method comprising:(a) providing a reaction volume at least partly delimited by at least one filtering barrier;(b) providing in the reaction volume at least one initiator nucleic acid and one templateindependent polymerase, wherein each initiator nucleic acid comprises a free 3 '-hydroxyl group;(c) performing a cycle comprising the steps of: i) providing in the reaction volume 3’-0 reversibly blocked nucleoside triphosphates under suitable conditions for polymerase-mediated extension of the at least one initiator nucleic acid, or of an extension product thereof, by incorporation of a 3’-O-reversibly blocked nucleoside triphosphate, resulting in the production of a reversibly blocked extension product; ii) deprotecting the 3’-O-reversibly blocked extension product, thereby forming an extension product having a free 3 ’-hydroxyl group ; and(d) repeating the cycle of step (c) until synthesis of the at least one polynucleotide is complete, wherein at step (c), the unincorporated 3’-O-reversibly blocked nucleoside triphosphates are removed from the reaction volume by diffusion through the filtering barrier, wherein the filtering barrier retains the at least one initiator nucleic acid and / or the templateindependent polymerase in the reaction volume.

2. The method of claim 1, wherein the at least one initiator nucleic acid and / or the template-independent polymerase is (are) in solution within the reaction volume.

3. The method of claim 1 or 2, wherein the template-independent polymerase is in solution within the reaction volume, and wherein the filtering barrier retains the templateindependent polymerase in the reaction volume.

4. The method of any one of claims 1 to 3, wherein the at least one initiator nucleic acid is in solution within the reaction volume, and wherein the filtering barrier retains the at least one initiator nucleic acid in the reaction volume.

5. The method of any one of claims 1 to 4, wherein the filtering barrier is a semi- permeable membrane, in particular selected from a microfiltration, ultrafiltration, nanofiltration and reverse osmosis membrane.

6. The method of any one of claims 1 to 5, wherein the filtering barrier is a molecular weight cut-off membrane.

7. The method of claim 6, wherein the membrane has a Molecular weight cutoff (MWCO) which is higher than the molecular weight of the 3’-O-blocked nucleoside triphosphate and lower than the molecular weight of the at least one initiator nucleic acid and / or the molecular weight of the template-independent polymerase.

8. The method of any one of claims 1 to 7, wherein the at least one initiator nucleic acid and / or the template-independent polymerase is (are) in solution within the reaction volume, wherein the filtering barrier is a molecular weight cut-off membrane and wherein the membrane has a Molecular weight cutoff (MWCO) which is higher than the molecular weight of the 3’-O-blocked nucleoside triphosphate and lower than the molecular weight of the at least one initiator nucleic acid and / or the molecular weight of the template-independent polymerase.

9. The method of any one of claims 6 to 8, wherein the membrane has a Molecular weight cutoff (MWCO) comprised between 1 and 55 kDa, in particular between 2 and 30 kDa, more particularly between 3 and 10 kDa, still more particularly between 5 and 10 kDa, most particularly between 7 and 10 kDa.

10. The method of any one of claims 1 to 9, wherein the filtering barrier is selected from a polymeric membrane, an inorganic membrane or a mixed polymeric-inorganic membrane.

11. The method of any one of claims 1 to 10, wherein the filtering barrier is made of a material selected from cellulose acetate, regenerated cellulose, polyethersulfone, polytetrafluoroethylene, polyvinylidene difluoride, polyethylene, polypropylene, polysulfone, polybenzimidazole, polyetheretherketone, polyacrylonitrile, polyamide, polyimide, polyetherimide, polyaniline, polypyrrole and mixtures thereof.

12. The method of any one of claims 1 to 11, wherein the unincorporated 3’-0 reversibly blocked nucleoside triphosphate are removed from the reaction volume by diffusing through the filtering barrier between step i) and step ii) of step (c) or after step ii) of step (c).

13. The method of any one of claims 1 to 12, wherein no template-independent polymerase is added in the reaction volume for at least 2 consecutive cycles of step (c).

14. The method of any one of claims 1 to 13, wherein the step of deprotecting the 3’- O-reversibly blocked extension product comprises providing a deblocking reagent which reacts with the 3’-O-protecting group of the 3’-O-reversibly blocked extension product.

15. The method of any one of claims 1 to 14, wherein the deblocking reagent is supplied in and / or removed from the reaction volume by diffusion through the filtering barrier.

16. The method of any one of claims 1 to 15, wherein the at least one initiator nucleic acid comprises at least one cleavable group, in particular wherein after step (d), the method comprises a further step of cleaving said at least one cleavable group, thereby releasing the at least one polynucleotide from the at least one initiator nucleic acid.

17. The method of any one of claims 1 to 16, wherein the at least one initiator nucleic acid comprises at least one capture tag, in particular wherein the method comprises a further step of capturing the at least one polynucleotide through binding of the capture tag.

18. The method of any one of claims 1 to 17, wherein the quantity of initiator nucleic acid in the reaction volume is comprised between 0.01 nmol and 100 nmol and / or wherein the concentration of the initiator nucleic acid in the reaction volume is comprised between IpM and 100 mM.

19. A device to enzymatically synthesize at least one polynucleotide, in particular according to the method of any one of claims 1 to 18, the device comprising a reaction volume at least partly delimited by a filtering barrier, wherein the device optionally comprises one or more means for supplying controlled amounts of reagents for synthesis of polynucleotides to the reaction volume, one or more of means for controlling temperature within the reaction volume, and one or more reservoirs for storing reagents for synthesis of polynucleotides.

20. A kit to enzymatically synthesize at least one polynucleotide, in particular according to the method of any one of claims 1 to 19, wherein said kit comprises a device according to claim 20 and one or more reagents for synthesis of polynucleotides selected from one or more initiator nucleic acids, 3’-0 reversibly blocked nucleoside triphosphates, template-independent polymerase and, optionally, reagents and buffers for the synthesis of polynucleotides such as a deblocking buffer.

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