Template-independent DNA synthesis using abortive infection system reverse transriptase

Abstract

The present invention provides a method for non-template dependent synthesis of oligo- and polynucleotides, which comprises immobilization of an Abi enzyme to facilitate the formation of a growing ssDNA by subsequent contacting with nucleotide triphosphates and final release of the oligo- or polynucleotide.

Description

[0001] Template-independent DNA synthesis using abortive infection system reverse transriptase

[0002] Technical field of the invention

[0003] The present invention relates to the field of recombinant DNA, oligo- and poly-nucleotides and biotechnology. Particularly, the present invention relates to methods for synthesis of oligonucleotides and polynucleotides with a desired sequence, without the use of a template, using abortive infection system reverse transcriptase, such as the enzyme AbiK from Lactococcus lactis or its derivatives.

[0004] Background of the invention

[0005] Synthetic DNA of a desired sequence is of increasing demand in different research and commercial sectors: data storage on DNA, molecular biology engineering approaches, nanotechnology, etc. With recent development of methods able to read DNA sequences, the capability to write DNA has lagged behind.

[0006] More than 40 years ago, phosphoramidite oligonucleotide sythesis (POS) was introduced which set the methodology for the first automated DNA synthesizer (Applied Biosystems) using solid-phase DNA synthesis which is used even today (1). This approach uses stepwise addition of building blocks derived from 5'-protected dimethoxytrtyl nucleotide phosphoramides in organic solvents to generate de novo DNA sequences in the absence of the template strand. The DNA synthesis industry wishes to enhance the efficiency of POS, which for instance shows inability to synthesize poly-repeat sequences, it is too slow as it takes ~10 minutes per nucelotide addition step and it involves the use of expensive chemical reagents and organic solvents which have adverse effect on nature. In recent years enzymatic DNA synthesis exploiting terminal deoxynucleotide transferase (TdT) was introduced for non-template de novo enzymatic DNA synthesis (TiEOS) and is integrated into commercial TiEOS methods (Camena Bioscience, Molecular Assemblies, DNA Script, ANSA Biotechnologies, Nuclera Nucleics). Enzyme TdT adds nucleotides (NTPs) to the 3'- accepting any of the four NTPs. To control the incorporation of the NTPs, reversible termination NTPs are applied ensuring the addition of a single NTP per reaction step. Modified NTPs with a reversible synthesisblocking terminator at 3' position of NTP are used i.e.: -3'-azidomethyl-protected NTPs by Nuclera Nucleics, Molecular Assemblies, 3'-ONH2-protected NTPs by DNA Script and 3'-O-2-nitrobenzyl by Camena Bioscience. These TiEOS approaches use the principles of solid-phase synthesis, as resin beads are pre-loaded with chemically synthesized single-stranded initiator DNA onto which TdT ligates 3'- protected NTP. Deblocking and washing steps are performed at each cycle before the next elongation step. Assembled DNA is released from the resin via a deoxyuridine enzymatic (uracyl DNA glycosilase) cleavage site integrated into initiator DNA. Such approach produces sideproducts and surplus reagents. An alternative approach ANSA Biotechnologies tether TdT to the base of an incoming NTP via cleavable linker to prevent the formation of homopolymeric nucleotide tracts, which makes the method expensive. Cleaving the linker enables the elongation steps. The above described TiEOS rely on fewer steps per synthesis cycle than POS, but still do not achieve quantitative elongations. Furthermore, the efficiency of the DNA synthesis by using TdT is reduced if the strand under construction begins to form secondary structures, which acts as a steric shield that prevent such double-stranded DNA to access the active site of TdT. Due to these detrimental impacts on DNA synthesis using TdT, new enzymes for TiEOS are needed to be explored. Several companies developed high-fidelity TdT enzymes (Camena Bioscience) or try to use TdT engineering to stabilize the enzyme (DNA Script, Nuclera nucleics, Molecular Assemblies).

[0007] Hence, there remains a need for more effective methods for preparing oligo and polynucleotides of a desired sequence for use in research and commercial sectors such as data storage on DNA, molecular biology engineering approaches etc. Specifically, there is a need for rapid methods to prepare smaller oligo and polynucleotides which can be used for encoding information as part of data storage, and which are sufficiently effective.

[0008] Summary of the invention

[0009] The object of the present invention is to overcome certain drawbacks in the existing methods for preparing oligo- and polynucleotides having a desired sequence, and to provide compositions useful for such methods.

[0010] Thus, in a first aspect the present invention provides a method for making one or more oligo- or polynucleotides comprising the steps:

[0011] (a) Optionally combining a nucleotide triphosphate(s), one or more cations, and an abortive infection system reverse transcriptase (Abi) enzyme which is immobilized at a solid surface, to facilitate a reaction to attach the nucleotide covalently to the Abi enzyme, such as to form a nucleotide-Abi enzyme conjugate, (b) Combining a nucleotide triphosphate(s), one or more cations, and an Abi enzyme- nucleotide conjugate which is immobilized at a solid surface, to facilitate a reaction to add the nucleotide into a growing ssDNA covalently attached to the Abi enzyme,

[0012] (c) Repeating step (b) until the one or more oligo- or polynucleotides is / are formed;

[0013] (d) Applying means to release the one or more oligo- or polynucleotides off said solid surface; such as to obtain said one or more oligo- or polynucleotides.

[0014] In an embodiment the Abi enzyme is AbiK from Lactocuccus lactis or a functional derivative thereof, such as a protein carrying an Abi-type, functional reverse transcriptase domain, exemplified by the AbiK reverse transcriptase domain (PROSITE entry PS50878), encompassing residues 1 to 326 of SEQ ID NO:1. In an embodiment the Abi enzyme is AbiA from Lactocuccus lactis (SEQ ID NO:4).

[0015] In a second aspect the present invention provides a composition comprising a collection of several oligo- or polynucleotides which all encode or display the alternating segments of nucleotides when a stretch of identical nucleotides is considered as encoding or displaying the particular nucleotide once, or the alternating segments containing only three of the four nucleotides and the absent nucleotide in a segment is considered as encoding or displaying the information.

[0016] In a third aspect the present invention provides a composition comprising a solid surface whereupon an Abi enzyme is immobilized in at least one location.

[0017] In a fourth aspect the present invention provides a method for regenerating the composition comprising a solid surface whereupon an Abi enzyme is immobilized in at least one location, comprising contacting of the at least one location of said composition with a solution comprising a nuclease, such as benzonase.

[0018] The present invention demonstrates the use of the AbiK enzyme of Lactococcus lactis for the production of protein-primed, template-independent single stranded DNA (ssDNA) carrying ordered segments composed of selected nucleotides. Such synthetic DNA can be used for data storage. AbiK protein is classified as the reverse transcriptase which provides phage resistance by polymerizing untemplated DNA. In particular, the method of the present invention is based on the Abi enzyme being immobilized on solid surface (e.g. surface plasmon resonance (SPR) chip) to de novo synthesize oligo- or polynucleotides, without the use of a nucleic acid template. The present invention uses unmodified tri-phosphate nucleotides which are e.g. injected (e. g. in microfluidics system of the SPR apparatus) over the Abi enzyme in a buffer. In the embodiment where a microfluidics system of a SPR apparatus is used, it is easy to control the injection time, volume, concentration of the ligand and analyte, buffer composition, and to select the temperature. The DNA product is covalently bound to the Abi enzyme during synthesis. The invention provides also the approach to release the synthetized DNA off the Abi enzyme on the solid surface.

[0019] The method of the present invention represents a major improvement over the known methods in that it provides faster, cheaper, and better controlled method for synthetic DNA synthesis that can be used e.g. for the data storage.

[0020] In addition, the synthesized DNA can be stored directly on the SPR chip at -20°C. When needed, the ssDNA can be removed from the chip by proteolytic degadation of AbiK, second strand produced by Klenow fragment and the DNA directly sequenced by Nanopore sequening.

[0021] Furthemore, the invention demonstrates that besides AbiK, of the three well-studied Abi reverse transcriptases, namely AbiK, AbiA and Abi-P2, also AbiA protein of Lactococcus lactis immobilized to the solid support is functional and can incorporate selected nucleotides.

[0022] Further features and advantages of the present invention are presented in the following text, drawings and claims thereof.

[0023] The present invention provides and is further characterized by the following items:

[0024] 1. Method for making one or more oligo- or polynucleotides comprising the steps:

[0025] (a) Optionally combining a nucleotide triphosphate(s), one or more cations, and an abortive infection system reverse transcriptase (Abi) enzyme which is immobilized at a solid surface, to facilitate a reaction to attach the nucleotide covalently to the Abi enzyme, such as to form a nucleotide-Abi enzyme conjugate,

[0026] (b) Combining a nucleotide triphosphate(s), one or more cations, and an Abi enzyme- nucleotide conjugate which is immobilized at a solid surface, to facilitate a reaction to add the nucleotide into a growing ssDNA covalently attached to the Abi enzyme,

[0027] (c) Repeating step (b) until the one or more oligo- or polynucleotides is / are formed;

[0028] (d) Applying means to release the one or more oligo- or polynucleotides off said solid surface; such as to obtain said one or more oligo- or polynucleotides. 2. The method according to item 1, wherein step(a) is performed once and step (b) is performed at least two times.

[0029] 3. The method according to item 1, wherein step (a) is not performed.

[0030] 4. The method according to any one of the preceding items, wherein said Abi enzyme is AbiK from Lactococcus lactis or a functional derivative thereof, such as a protein carrying an Abi-type, functional reverse transcriptase domain, exemplified by the AbiK reverse transcriptase domain (PROSITE entry PS50878), encompassing residues 1 to 326 of SEQ ID NO:1.

[0031] 5. The method according to any one of the preceding items wherein said Abi enzyme is a polypeptide having at least 80% sequence identity to SEQ ID NO:1, at least 85% sequence identity to SEQ ID NO:1, at least 90% sequence identity to SEQ ID NO:1, at least 95% sequence identity to SEQ ID NO:1, at least 98% sequence identity to SEQ ID NO:1.

[0032] 6. The method according to any one of the preceding items, wherein said AbiK protein from Lactococcus lactis or a functional derivative thereof is a protein having at least 85% identity to SEQ ID NO: 1.

[0033] 7. The method according to any one of the preceding items, wherein said AbiK protein from Lactococcus lactis or a functional derivative thereof is a protein having at least 95% identity to SEQ ID NO: 1.

[0034] 8. The method according to any one of the preceding items, wherein said AbiK protein from Lactococcus lactis or a functional derivative thereof is a truncated derivative of SEQ ID NO:1 exhibiting at least 85% identity or at least 90% identity to the corresponding part of SEQ ID NO:1.

[0035] 9. The method according to any one of the preceding items, wherein said Abi enzyme is AbiK from Lactococcus lactis, SEQ ID NO: 1.

[0036] 10. The method according to any one of the preceding items, wherein said Abi enzyme is AbiK from Lactococcus lactis or AbiA from Lactococcus lactis, or a protein exhibiting sequence homology to AbiK or AbiA.

[0037] 11. The method according to any one of items 1-3, wherein said Abi enzyme is AbiA from Lactococcus lactis or a functional derivative thereof. 12. The method according to any one of the preceding items, wherein said solid surface is a surface plasmon resonance (SPR) sensor chip.

[0038] 13. The method according to item 12, wherein the repetitions of step (b) is followed using by a surface plasmon resonance refractometer.

[0039] 14. The method according to item 12 or 13, wherein the timing of the repetitions of step (b) is controlled by a surface plasmon resonance refractometer.

[0040] 15. The method according to any one of the preceding items, wherein said nucleotide triphosphate is injected across the Abi enzyme to facilitate a reaction to add the nucleotide into a growing ssDNA covalently attached to the Abi enzyme.

[0041] 16. The method according to item 15, wherein said injection of nucleotide triphosphate is performed using a microfluidic system.

[0042] 17. The method according to item 15 or 16, wherein the reaction to add the nucleotide is being followed in real time in an automated set-up.

[0043] 18. The method according to any of the preceding items, wherein in steps (a) and (b) said one or more cations comprises Mn2* and / or Mg2+.

[0044] 19. The method according to any of the preceding items, wherein in steps (a) and (b) said nucleotide triphosphate, one or more cations, and an Abi enzyme or a functional derivative thereof are combined in a buffer comprising a surfactant such as polysorbate 20.

[0045] 20. The method according to any of the preceding items, wherein in steps (a) and (b) said nucleotide triphosphate, one or more cations, and an Abi enzyme are combined in a buffer comprising Tris, NaCI, MgCL, and a surfactant.

[0046] 21. The method according to any of the preceding items, wherein in step (a) said nucleotide triphosphate, one or more cations, and an Abi enzyme are combined in a buffer comprising 20 mM Tris pH 8.3, 140 mM NaCI, 2 mM MgCL, 0.005% polysorbate 20.

[0047] 22. The method according to any one of the preceding items, wherein in step (c) said means to release the one or more oligo- or polynucleotides off said solid surface is selected from one or more from the group consisting of a suitable restriction enzyme, an oligonucleotide, a protease and a reagent, such as 20 mM Tris-HCI (pH 8.8 at 25°C), 10 mM (NH^SC , 10 mM KCI, 0,1 mg / mL BSA, 1% (v / v) Triton X-100, 2 mM MgSO4.

[0048] 23. The method according to any one of the preceding items, wherein said means to release the one or more oligo- or polynucleotides is proteinase K.

[0049] 24. The method according to any one of the preceding items, wherein said means to release the one or more oligo- or polynucleotides is USER enzyme (Uracil-specific excision reagent (USER) comprising an enzyme mix of uracil DNA glycosylase and DNA glycosylase-lyase).

[0050] 25. The method according to any one of the preceding items, further comprising a step wherein the released oligo- or polynucleotide ssDNA in treated with one or more restriction enzymes to remove contaminating DNA.

[0051] 26. The method according to any one of the preceding items, wherein said nucleotide triphosphate is selected from the group consisting of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxyuridine triphosphate (dUTP).

[0052] 27. The method according to any one of the preceding items, wherein said oligo- or polynucleotide contains from 5 to 200 nucleobases, from 5 to 100 nucleobases, from 5 to 50 nucleobases, from 5 to 30 nucleobases, from 20 to 200 nucleobases, from 20 to 100 nucleobases, from 20 to 50 nucleobases, or from 5 to several hundred nucleobases.

[0053] 28. Composition comprising a collection of several oligo- or polynucleotides which all encode or display the alternating segments of nucleotides when a stretch of the alternating segments containing only three of the four nucleotides and the absent nucleotide in a segment is considered as encoding or displaying the information.

[0054] 29. The method according to any one of the claims, wherein said Abi enzyme is AbiA from Lactococcus lactis, SEQ. ID NO: 4.

[0055] 30. Composition comprising a solid surface whereupon an Abi enzyme is immobilized in at least one location.

[0056] 31. The composition according to item 30, wherein said Abi enzyme is AbiK from Lactococcus lactis or a functional derivative thereof. 32. The composition according to item 30, wherein said Abi enzyme is AbiA from Lactococcus lactis or a functional derivative thereof.

[0057] 33. The composition according to any one of items 30-32, wherein said solid surface is a surface plasmon resonance (SPR) sensor chip.

[0058] 34. The composition according to any one of items 30-33, wherein said Abi enzyme is immobilized to said solid surface by amine-coupling.

[0059] 35. The composition according to any one of the items 30-34, wherein said immobilization is by (i) surface activation by l-ethyl-3-(3-dimethylpropyl)-carbodiimide (EDC) and N- hydroxysuccinimide (NHS), (ii) coupling of the Abi enzyme, and (iii) surface blocking by ethanol amine.

[0060] 36. The composition according to any one of items 30-33, wherein said solid surface is covered by oligonucleotides comprising from 10-30 identical nucleotides, and said Abi enzyme has been incubated with the nucleotide triphosphate of the matching nucleotides, such that immobilization is by way of hybridization between the oligonucleotides on said solid surface and the oligonucleotide formed by the Abi enzyme and attached thereto.

[0061] 37. The composition according to item 36, wherein said solid surface is covered by (C)I5-25 (oligonucleotide having from 15 to 25 cytosines) and the Abi enzyme is incubated with dGTP.

[0062] 38. Method for regenerating the composition as defined in any of items 30-35, comprising contacting of the at least one location of said composition with a solution comprising a nuclease, such as benzonase.

[0063] Brief description of the figures

[0064] FIG 1. Schematic representation of the procedure for using the surface plasmon resonance refractometer (prepared using BioRender, https: / / www.biorender.com / ).

[0065] FIG 2. The amine-coupling immobilization profile of AbiK on the CM5 chip: (i) surface activation by EDC / NHS, (ii) AbiK coupling, and (iii) surface blocking by ethanol amine. AbiK was injected at 50 pg / ml in sodium acetate buffer (pH 5.0). The 21160 response units (RU) and 18150 RU of AbiK were immobilized to the flow cell 2 (Fc2) and 3 (Fc4) of the presented experiment, respectively. FIG. 3. Sensorgrams are showing the integration of the deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), or deoxyguanosine triphosphate (dGTP) by the AbiK enzyme immobilized to the surface of the CM5 chip to approximately 20000 RU (set as 0 RU) at temperature 25°C. Nucleotides were injected at the indicated time points at 100 pM concentration for 200 s at flow 10 pl / min. The regeneration of the AbiK-sensor chip surface was performed by the nuclease benzonase which degradates and removes the produced DNA off the sensor chip. Shown is the 600 s injection of 20 enzyme units (U) of benzonase. Nucleotides were injected at 100 pM concentration. The experiments were performed in the running buffer 20 mM Tris pH 8.3, 140 mM NaCI, 0.005% P20 at flow 10 pl / min.

[0066] FIG. 4. Sensorgrams of the temperature-dependent activity of chip immobilized AbiK (immobilized in sodium acetate buffer pH 5.0 to ~13325 RU to the F4, set as RU 0). Sensorgrams show the integration of the deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), or deoxyguanosine triphosphate (dGTP) in the growing single stranded DNA at temperatures ranging from 10°C to 35°C. At time point 1100 seconds, a pulse of dNTPs was injected to show that saturation of AbiK with nucleotides was not reached. After dNTP injection (at ~1500 seconds) nuclease benzonase as injected to regenerate the AbiK-chip surface (not presented on sensorgrams). Each individual nucleotide, either alone or in the mixture of nucleotides, was injected at 100 pM concentration. The experiments were performed in the running buffer 20 mM Tris pH 8.3, 140 mM NaCI, 0.005% P20 at flow 10 pl / min.

[0067] FIG. 5. Sensorgram of injection of selected dNTP over the chip immobilized AbiK (AbiK was immobilized at pH 5.0 to 21000 RU, sensorgram starting at time 0 s). The dNTPs were injected at 100 pM concentration for 90 s at flow 10 pl / min. At the indicated timepoints oligonucleotides (18 nucleotide) carrying poly A (5'-AAAAAAAAAAAAAAAAAA-3', SEQ ID NO:16), poly C (5'- CCCCCCCCCCCCCCCCCC-3', SEQ ID NO: 17) or poly T (5 -TTI I I I I I I I I I I I I I I T-3', SEQ ID NO: 18) were sequentially injected over the synthesised DNA by AbiK. The sensorgram on the top shows complete cycle and the indicated parts of sensorgrams are enlarged in panels A-D. The experiment was performed in the running buffer 20 mM Tris pH 8.3, 140 mM NaCI, 2 mM MgCL, 0.005% P20 at flow 10 pl / min.

[0068] FIG. 6. Sensorgram of injection of 10 pM dNTPs or 1 pM deoxyuridine triphosphate (dUTP), 100 pM dGTP, or 100 pM dATP over the chip immobilized AbiK (AbiK was immobilized at pH 5.0 to 21000 RU, sensorgram starting at time 0 s). At indicated timepoints oligonucleotides (18 nucleotide) carrying poly A (5'-AAAAAAAAAAAAAAAAAA-3'), poly C (5'-CCCCCCCCCCCCCCCCCC-3') or poly T (5'-TT l l l l l l l l l l l l l l TT-3') were sequentially injected over the AbiK and the synthesised DNA. The sensorgram on the top shows complete cycle and the indicated parts of sensorgrams are enlarged in panels A and B. The experiment was performed in the running buffer 20 mM Tris pH 8.3, 140 mM NaCI, 2mM MgCL, 0.005% P20. In panel B the flow rates of the running buffer are shown. In panel B also, the injection of the USER Enzyme that cleaves DNA at uridines is shown. USER enzyme was injected at the flow rate of 5 pl / min for 800 s at 20U. The enzyme caused the cleavage of approximately 400 RU of the DNA off the AbiK immobilized on the SPR chip.

[0069] FIG. 7. Sensorgram of injections of 100 pM dCTP, dGTP, dTTP, or dATP across the chip immobilized AbiK (AbiK was immobilized at pH 5.0 to approximately 14000 RU, to Fc2, Fc3, or Fc4 (flow cell); sensorgram starting at time 0 s, response set to 0 RU). The experiment was performed in the running buffer 20 mM Tris pH 8.3, 140 mM NaCI, 2mM MgCL, 0.005% P20. The nucleotide and the time of each injection is shown below the sensorgrams. The experiment was performed at flow 10 pl / min.

[0070] FIG. 8. Workflow for the polymerisation of dGTP, dTTP, or dATP into the selected ssDNA molecule, followed by the release of ssDNA from AbiK by proteinase K treatment and proceeded by the temperature inactivation of the protease, degradation of the environmental DNA by selected restriction enzymes, and amplication and sequencing of the AbiK-polymerized synthetic DNA (prepared using Biorender).

[0071] FIG. 9. Sensorgram of injections of 100 pM dGTP, dTTP, or dATP or the mixture of the three nucleotides across the chip immobilized AbiK (AbiK was immobilized at pH 5.0 to approximately 19000 RU, to Fcl, Fc2, Fc3, or Fc4 (flow cell); sensorgram starting at time 0 s, response set to 0 RU; results presented only for the Fc2). The experiment was performed in the running buffer 20 mM Tris pH 8.3, 140 mM NaCI, 2 mM MgCL, 0.005% P20. The nucleotide and the time of each injection is shown below the sensorgrams. "Mix" denotes injection of mixture of 100 pM dATP (A), 100 pM dGTP (G), and 100 pM dTTP (T). The experiment was performed at flow 20 pl / min.

[0072] FIG. 10. Sensorgram of injections of 100 pM dCTP, dATP, dTTP, or dGTP across the chip immobilized AbiK (AbiK was immobilized at pH 5.0 to approximately 20000 RU, to Fcl, Fc2, Fc3, or Fc4 (flow cell); sensorgram starting at time 0 s, response set to 0 RU; results presented only for the Fc2). The experiment was performed in the running buffer 20 mM Tris pH 8.3, 140 mM NaCI, 2 mM MgCL, 0.005% P20. The nucleotide and the time of each injection is shown below the sensorgrams. The experiment was performed at flow 20 pl / min.

[0073] FIG. 11. Schematic illustration of the attachment of AbiK protein (subunits shown as cylinders) to the single stranded DNA carrying 20 cytosines, which is attached to the solid support via biotin (Bio) - streptavidin interaction. Prior to loading, AbiK is incubated with 100 pM dGTP in the buffer containing 20 mM Tris pH 8.3, 140 mM NaCI, 2 mM MgCL, 0.005% P20, to generate a short stretch of ssDNA rich in guanines. This enables AbiK-DNA nucleoprotein complex to hybridize to the oligonucleotide containing cytosines (enlarged). This interaction can be reversed by incubation of the surface with 10-50 mM NaOH for few seconds, which destroys the C-G hydrogen bonds. (TEG - tetrethylene glycol).

[0074] FIG. 12. Schematic representation of the workflow depicting the synthesis of DNA with the sequence encoding the word "DNA" twice, the release of single-stranded DNA (ssDNA) from AbiK by proteinase K treatment followed by the temperature inactivation of the protease, the synthesis of the second DNA strand by the Klenow fragment, temperature inactivation of Klenow fragment, and nanopore sequencing of the synthesized DNA (created with BioRender, https: / / www.biorender.com / ). b, Sensorgram depicting the polymerization of ssDNA encoding "DNA" and "DNA", followed by the injection of dNTPs or dGTP (G). Four different nucleotide mixtures containing three of the four nucleotides (dATP, dTTP, dGTP, or dCTP; mixtures: ACG, TCG, ATC, or ATG) were injected over immobilized AbiK ("'20000 RU) in a sequence encoding the word "DNA." A dilution of the single nucleotide dGTP was injected in the middle and at the end of the custom sequence. A mixture of all four nucleotides (dNTPs) was injected at the beginning and end of the experiment. All nucleotides or nucleotide mixtures were injected over AbiK at a concentration of 100 pM and a flow rate of 40 pL / min for the number of seconds indicated under the marked nucleotide or nucleotide mixture in the sensorgram. The experiment was performed in a running buffer containing 20 mM Tris (pH 8.3), 140 mM NaCI, 2 mM MgCL, and 0.005% P20. The rectangles on the sensorgram represent selected sequences of the polymerized DNA.

[0075] FIG. 13. Sensorgrams are showing the integration of the deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), or deoxyuridine triphosphate (dUTP) in the single stranded DNA by the AbiA or the AbiK enzyme immobilized to the surface of the CM5 chip. The experiments were performed at temperature 25°C. The AbiK was immobilized to the flow cell 2 to approximately 6500 RU and the AbiA was immobilized to the flow cell 4 to approximately 4700 RU (responses set as 0 RU). Responses measured on the flow cell 2 are marked by AbiK and the response measured on the flow cell 4 by the AbiA. Nucleotides were injected at the indicated time points at 100 pM concentration for 60 s at flow 10 pl / min. In addition, at the indicated time point a mixture of dATP, dGTP, dCTP, dTTP (each at 100 pM) was injected. The experiments were performed in the running buffer 20 mM Tris pH 8.3, 2 mM MgCI2, 140 mM NaCI, 0.005% P20 at flow 10 pl / min.

[0076] FIG. 14. Sensorgrams are showing the integration of the deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), or deoxyuridine triphosphate (dUTP) in the single stranded DNA by the AbiA or the AbiK enzyme immobilized to the surface of the CM5 chip. The experiments were performed at temperature 25°C. The AbiK was immobilized to the flow cell 2 to approximately 6500 RU and the AbiA was immobilized to the flow cell 4 to approximately 4700 RU (responses set as 0 RU). Responses measured on the flow cell 2 are marked by AbiK and the response measured on the flow cell 4 by the AbiA. Nucleotides were injected at the indicated time points at 100 pM concentration for 60 s at flow 10 pl / min. In addition, at the indicated time points a mixture of dATP, dGTP, dCTP, dTTP (each at 100 pM) was injected. The experiments were performed in the running buffer 20 mM Tris pH 8.3, 2 mM MnCI2, 140 mM NaCI, 0.005% P20 at flow 10 pl / min.

[0077] Detailed description of the invention

[0078] In recent years, for the template-independent enzymatic oligonucleotide synthesis (TiEOS), the enzyme terminal deoxynucleotide transferase (TdT) is the preferred enzyme. The native or modified enzyme TdT were shown to enable de novo synthesis of oligo or polynucleotides in controlled manner, preventing the subsequential coupling of the next nucleotide until further treatment, by using stepwise addition of modified-protected nucleotides or nucleotide-enzyme conjugates, in the absence of a template, reviewed in (1-3). One approach uses modified TdT, solid substrate for retaining the polynucleotide during synthesis, an initiator strand of more than 3 nucleotides, and reversible terminal nucleotides for controlled addition of selected nucleotides in the growing ssDNA polymer. The terminating group on the modified nucleotide pauses synthesis with the addition of each new base, whereupon the terminal group is cleaved, which enables the synthesis to proceed. In 2018, a report was published on DNA synthesis using TdT bound to a single dNTP via a reversible linker. In this method, DNA synthesis is controlled by the release of the TdT enzyme, after the enzyme has added the attached nucleotide, from the 3'- of the growing ssDNA by de-crosslinking TdT-ssDNA conjugate (4). Another approach utilizes the competition between the enzyme apyrase and TdT (5). The apyrase in the reaction mixtures degrades the nucleotides, limiting the number of nucleotides with the same base in the growing ssDNA chain. An approach that would enable the use of standard nucleotides and would govern a robust, custom, nontemplate DNA synthesis, would provide a much-needed approach for the enhanced DNA synthesis technology.

[0079] Among prokaryotic reverse transcriptases are also abortive infection (Abi) reverse transcriptases, with well-studied proteins: AbiK, AbiA, and Abi-P2. In 2005 it was described that the abiK gene of Lactococcus lactis is a part of an operon that includes two additional open reading frames and has antiphage activity (6). The AbiK protein is a protein of 599 amino acids with molecular size of 71.4 kDa and a pl of 7.98 (7). AbiK was shown to be able to polymerise long, random sequence single stranded DNAs (8). It has been observed that AbiK has preference of dATP > dTTP or dCTP or dGTP for incorporation into DNA polymer (8). AbiK was shown to be a hexameric protein and that such oligomerisation is essential for the DNA polymerization (9). The structure of the AbiK-DNA adducts reveals interactions between the DNA and the active site, as well as covalent attachment to a tyrosine residue used for protein priming.

[0080] The AbiK protein has not yet been described to be capable of non-template, ordered oligo or polynucleotide synthesis.

[0081] The method.

[0082] In a first aspect the present invention provides a method for making one or more oligo- or polynucleotides comprising the steps:

[0083] (a) Optionally combining a nucleotide triphosphate(s), one or more cations, and an abortive infection system reverse transcriptase (Abi) enzyme which is immobilized at a solid surface, to facilitate a reaction to attach the nucleotide covalently to the Abi enzyme, such as to form a nucleotide-Abi enzyme conjugate,

[0084] (b) Combining a nucleotide triphosphate(s), one or more cations, and an Abi enzyme- nucleotide conjugate which is immobilized at a solid surface, to facilitate a reaction to add the nucleotide into a growing ssDNA covalently attached to the Abi enzyme,

[0085] (c) Repeating step (b) until the one or more oligo- or polynucleotides is / are formed;

[0086] (d) Applying means to release the one or more oligo- or polynucleotides off said solid surface; such as to obtain said one or more oligo- or polynucleotides.

[0087] In one embodiment step (a) is performed once and step (b) is performed at least two times. In another embodiment step (a) is not performed.

[0088] In another embodiment the Abi enzyme is from Lactococcus lactis. Some of the different Abi enzyme from Lactococcus lactis are described in e.g. (7).

[0089] In another embodiment the Abi enzyme is AbiK from Lactococcus lactis or a functional derivative thereof.

[0090] In yet another embodiment the Abi enzyme is a polypeptide having at least 80% sequence identity to SEQ ID NO:1, at least 85% sequence identity to SEQ ID NO:1, at least 90% sequence identity to SEQ ID NO:1, at least 95% sequence identity to SEQ ID NO:1, at least 98% sequence identity to SEQ ID NO:1.

[0091] In another embodiment the AbiK protein from Lactococcus lactis or a functional derivative thereof is a protein having at least 85% identity to SEQ ID NO: 1.

[0092] In another embodiment the AbiK protein from Lactococcus lactis or a functional derivative thereof is a protein having at least 95% identity to SEQ ID NO: 1.

[0093] In another embodiment the AbiK protein from Lactococcus lactis or a functional derivative thereof is a truncated derivative of SEQ ID NO:1 exhibiting at least 85% identity or at least 90% identity to the corresponding part of SEQ ID NO:1.

[0094] In another embodiment the Abi enzyme is AbiK from Lactococcus lactis, SEQ ID NO: 1.

[0095] In another embodiment the Abi enzyme is AbiK from Lactococcus lactis or AbiA from Lactococcus lactis, or a protein exhibiting sequence homology to AbiK or AbiA.

[0096] In another embodiment the Abi enzyme is AbiA from Lactococcus lactis or a functional derivative thereof.

[0097] In another embodiment the solid surface is a surface plasmon resonance (SPR) sensor chip.

[0098] In another embodiment the repetitions of step (b) is followed using by a surface plasmon resonance refractometer. In another embodiment the timing of the repetitions of step (b) is controlled by a surface plasmon resonance refractometer.

[0099] In another embodiment the nucleotide triphosphate is injected across the Abi enzyme to facilitate a reaction to add the nucleotide into a growing ssDNA covalently attached to the Abi enzyme.

[0100] In another embodiment the injection of nucleotide triphosphate is performed using a microfluidic system.

[0101] In another embodiment the reaction to add the nucleotide is being followed in real time in an automated set-up.

[0102] In another embodiment in steps (a) and (b) said one or more cations comprises Mn2* and / or Mg2+.

[0103] In another embodiment in steps (a) and (b) said nucleotide triphosphate, one or more cations, and an Abi enzyme or a functional derivative thereof are combined in a buffer comprising a surfactant such as polysorbate 20.

[0104] In another embodiment in steps (a) and (b) said nucleotide triphosphate, one or more cations, and an Abi enzyme are combined in a buffer comprising Tris, NaCI, MgCL, and a surfactant.

[0105] In another embodiment the in step (a) said nucleotide triphosphate, one or more cations, and an Abi enzyme are combined in a buffer comprising 20 mM Tris pH 8.3, 140 mM NaCI, 2 mM MgCL, 0.005% polysorbate 20.

[0106] In another embodiment the in step (c) said means to release the one or more oligo- or polynucleotides off said solid surface is selected from one or more from the group consisting of a suitable restriction enzyme, an oligonucleotide, a protease and a reagent, such as 20 mM Tris-HCI (pH 8.8 at 25°C), 10 mM (NH4)2SO4, 10 mM KCI, 0,1 mg / mL BSA, 1% (v / v) Triton X-100, 2 mM MgSO4.

[0107] In another embodiment the means to release the one or more oligo- or polynucleotides is proteinase K.

[0108] In another embodiment the means to release the one or more oligo- or polynucleotides is USER enzyme (Uracil-specific excision reagent (USER) comprising an enzyme mix of uracil DNAglycosylase and DNA glycosylase-lyase). In another embodiment the method further comprises a step wherein the released oligo- or polynucleotide ssDNA in treated with one or more restriction enzymes to remove contaminating DNA.

[0109] In another embodiment the nucleotide triphosphate is selected from the group consisting of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxyuridine triphosphate (dUTP).

[0110] In another embodiment the oligo- or polynucleotide contains from 5 to 200 nucleobases, from 5 to 100 nucleobases, from 5 to 50 nucleobases, from 5 to 30 nucleobases, from 20 to 200 nucleobases, from 20 to 100 nucleobases, from 20 to 50 nucleobases, or from 5 to several houndred of nucleobases.

[0111] Compositions.

[0112] In a second aspect the present invention provides a composition comprising a collection of several oligo- or polynucleotides which all encode or display the alternating segments of nucleotides when a stretch of identical nucleotides is considered as encoding or displaying the particular nucleotide once, or the alternating segments containing only three of the four nucleotides and the absent nucleotide in a segment is considered as encoding or displaying the information.

[0113] In an embodiment the alternating sequence of nucleotides does not contain any two adjacent nucleotides which are identical.

[0114] In a third aspect the present invention provides a composition comprising a solid surface whereupon an Abi enzyme is immobilized in at least one location.

[0115] In another embodiment the solid surface is a surface plasmon resonance (SPR) sensor chip.

[0116] In another embodiment the Abi enzyme is immobilized to said solid surface by amine-coupling. In yet another embodiment the immobilization is by (i) surface activation by l-ethyl-3-(3- dimethylpropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), (ii) coupling of the Abi enzyme, and (iii) surface blocking by ethanol amine.

[0117] In another embodiment the solid surface is covered by oligonucleotides comprising from 10-30 identical nucleotides, and said Abi enzyme has been incubated with the nucleotide triphosphate of the matching nucleotides, such that immobilization is by way of hybridization between the oligonucleotides on said solid surface and the oligonucleotide formed by the Abi enzyme and attached thereto.

[0118] In another embodiment the solid surface is covered by (C)I5-25 (oligonucleotide having from 15 to 25 cytosines) and the Abi enzyme is incubated with dGTP.

[0119] Regeneration of solid surface.

[0120] In a fourth aspect the present invention provides a method for regenerating the composition comprising a solid surface whereupon an Abi enzyme is immobilized in at least one location, comprising contacting of the at least one location of said composition with a solution comprising a nuclease, such as benzonase.

[0121] Definitions.

[0122] The term "functional derivative" as used herein in relation to an enzyme is intended to mean a modified enzyme having a physical structure resembling that of the enzyme, and which catalyzes the same reaction as the parent (unmodified) enzyme. For example, AbiK from Lactococcus lactis or a functional derivative thereof encompasses the AbiK from Lactococcus lactis and modified enzymes catalyzing the same reaction but having a slightly modified amino acid sequence, being a truncated version thereof or having one or more amino acid residues being non-natural, e.g. D- amino acids, Aib or amino acids comprising a chemically modified side chain. E. g. a truncated AbiK protein can carry only an Abi-type, functional reverse transcriptase domain, exemplified by the AbiK reverse transcriptase domain (PROSITE entry PS50878), encompassing residues 1 to 326 of SEQ ID NO:1.

[0123] The term percent "sequence identity" (% sequence identity) between two polypeptide or DNA sequences is determined from a pairwise sequence alignment created by using program "Needle" (as implemented in the European Molecular Biology Open Software Suite (EMBOSS), version 6.3.1.2 or later (Trends in Genetics (2000) 16 (6), p. 276-277) with applying a gap open penalty of 10, a gap extension penalty of 0.5, the matrix EBLOSUM62 (which is the "Needle" EMBOSS version of matrix BLOSUM62) for protein sequences, and the matrix EDNAFULL (which is the "Needle" EMBOSS version of matrix DNAFULL) for nucleotide sequences, by the following calculation: % sequence identity = (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment), which is also reported as "Longestjdentity" in the output of "Needle" EMBOSS when parameter nobrief" is applied. Program "Needle" EMBOSS has implemented the algorithm of Needleman &

[0124] Wunsch (J. Mol. Biol. (1979) 48, p. 443-453) for aligning of two sequences.

[0125] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

[0126] Examples

[0127] Example 1:

[0128] Here we use purified recombinant AbiK (9) and have immobilized it on the SPR chip to achieve ordered incorporation of nucleotides into segments consisting of selected deoxyribonucleotides (dNTPs). To do this, we used the injection system of the SPR apparatus to inject the selected dNTP over AbiK as a substrate, which incorporates dNTPs into the growing ssDNA chain covalently attached to the AbiK (FIGURE 1). Repeating this step and using a selected order of dNTPs, AbiK generates a single stranded DNA carrying selected (non-random) stretches of nucleotides without the need of the template DNA. In such synthetic DNA the information e.g. an alphabet can be encoded (bases from 5'- to -3': sequences of nucleotides carrying one of the adenosine, thymidine, cytosine or guanine base), see Table 1.

[0129] Table 1. Sequence of nucleotides encoding the letters of the alphabet.

[0130] The SPR measurements were performed at the Infrastructural Centre for Analysis of Molecular Interactions at the Department of Biology, University of Ljubljana on a Biacore T200 (GE Healthcare) at 25 °C. We isolated AbiK at concentration 2.7 mg / ml as described in a previous report (9) and dissolved it before immobilization to the CM5 chip (Cytiva) in sodium acetate buffer (pH 5.0) to the final concentration of 50 pg / ml. As shown in FIGURE 2, the surface of the CM5 sensor chip was activated with a 7-min injection of the mixture N-hydroxysuccinimide and l-ethyl-3-(3- dimethylpropyl)-carbodiimide (EDC / NHS) as suggested by the manufacturer. The 50 pg / ml AbiK diluted in 10 mM Na-acetate, pH 5.0, was applied on the flow cell 2 or flow cell 4. The first and third flow cell was left unmodified and served as a control for non-specific binding of tested compounds. The sensorgrams show in the results are the responses obtained on the Fes 3 or 4 referenced for the responses obtained on the Fes 1 or 2. The remaining active groups on the surface of both flow cells were deactivated with a 7-min injection of ethanolamine. The level of immobilized enzyme was around 20000 response units (marked in figure legends). The data were analysed with the Biacore T200 Evaluation Software (GE Healthare).

[0131] Either dNTP (deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), or deoxyguanosine triphosphate (dGTP)) was injected (~100 pM as indicated) in the running buffer composed of 20 mM Tris pH 8.3, 140 mM NaCI, 2 mM MgCL, 0,005% P20, separately, over the chip-immobilized AbiK. We tested the effect of temperature on the activity of the AbiK protein immobilized on the SPR chip (FIGURES 3 and 4). The polymerisation reactions were carried out at 10, 15, 20, 25, 30 or 35°C. Results show that: (i) chip-immobilized AbiK remains active and, according to the increasing responses upon nucleotide injections can incorporate either dATP, dTTP, dCTP, or dGTP into the growing single stranded chain, hence generating a strand of DNA with selected strands of A, T, G, or C sequence, (ii) the activity of the enzyme is temperature dependent. The results indicate that the enzyme is minimally active at temperatures ranging from 10-15°C and the enzyme activity increases at elevated temperatures tested.

[0132] Example 2: Release of oligonucleotide by Benzonase.

[0133] In FIGURE 3 we show that the nuclease Benzonase (Sigma, USA) can remove, cleave the DNA generated by the AbiK, and release the DNA from the surface of the SPR chip.

[0134] The results presented in FIGURES 5 and 6 show that by using AbiK immobilized on the CM5 chip, we can obtain a DNA strand carrying ordered segments of a selected nucleotide A, T, or G. To show this we used 18-mer oligonucleotides carrying adenosine, thymidine, or cytosine bases (Microsynth, Austria; the poly G sequence cannot be purchased). These results show that polyA, polyT, or polyC primers hybridize only to the DNA which is generated by the AbiK enzyme, thus after the injection of the selected nucleotide, which is incorporated by AbiK into the growing single stranded DNA. Furthermore, the results presented in FIGURE 6 show that the chip-immobilized AbiK can efficiently incorporate also deoxyuridine triphosphate (dUTP) into the ssDNA. This enables one to release the covalently attached ssDNA to the AbiK, containing uridines, from the surface of the sensor chip, using the USER enzyme (NEB, USA) which catalyzes the nucleotide gap at the location of the uracil residue. The released DNA can be collected in the elution volume coming from the SPR apparatus, for the downstream analyses and applications.

[0135] Example 3: Incorporation of nucleotides in multiple cycles into growing ssDNA.

[0136] In addition, we sequentially injected dATP, dTTP, dCTP, or dGTP across the AbiK, which was immobilizied on the CM5 chip to show, that AbiK can incorporate nucleotides in multiple cycles into the growing ssDNA (FIGURE 7). This can be observed from the increase of the response units (RU) upon injection of the selected one of the four nucleotides.

[0137] To directly show that AbiK can be used to catalyse the synthesis of the ssDNA with specific nucleotide sequence, we (FIGURE 8): (i) immobilized AbiK on four flow cells of the SPR chip CM5; (ii) injected three nucleotides dATP or dTTP or dGTP (all at concentration of 100 pM ) or the mixture of dATP, dTTP, dGTP (all at concentration of 100 pM) at flow rate 20 pl / min and 25°C for selected injection time (10 - 200 s) across the chip-immobilized AbiK (it is of note, that we injected the mixture of dATP, dGTP, and dTTP to govern the annealing of random DNA primers used for PCR amplification of the synthetic DNA); (iii) ejected the SPR chip carrying the synthetic ssDNA covalently attached to AbiK from the SPR apparatus and we treated the surface of the chip by proteinase K (0.4 mg / mL in EquiPhi29™ DNA Polymerase buffer at 55°C for lh); (iv) afterwards, proteinase K was inactivated at 95°C for 15 min; (v) reaction mixture was cooled down and the fastdigest restriction enzymes Dpnl (target DNA sequence 5'- GATC-3'), Bspl43l (target DNA sequence 5'-GATC-3') and Alul (target DNA sequence 5'-AGCT-3'), all Thermo Fisher Scientific), were added at 0.2 U into the reaction mixture to degrade the DNA contaminations carrying nucleotides containing cytosine (37°C, 30 min); next (vi) the ssDNA polymerised by AbiK was amplified by PCR using Exo-Resistant Random Primers and Phusion DNA polymerase (both Thermo Fisher Scientific; according to manufacturer's instructions with annealing temperature of 55°C and 2 min of elongation time at 72°C for 35 cycles); and (vii) sequenced using Oxford Nanopore Technology, Full PlasmidSeq service provided by company Microsynth. As a negative control, in the control reaction mixture the water was used instead of the sample containing the synthesized DNA and the reaction was carried out as described in steps iii-viii.

[0138] Example 4: Synthesis of oligonucleotides having variation in some positions. Results in FIGURE 9 show the polymerisation of DNA composed of nucleotides carrying either nucleobase adenine, thymine, or guanine. We injected the nucleotides in the order (G - dGTP, A - dATP, T - dTTP) to obtain SEQ ID NO: 5: G-T-A-G-T-A-G-T-A-MIX(G. A, T)-G-T-A-G-T-A-G-T-A-MIX(G, A, T)-A-T-G-MIX(G, A, T)-3'.

[0139] By using nanopore sequencing we obtained more than 100 sequencing reads and approximately 50 sequencing reads for the ssDNA and the control sample, respectively. We checked the sequences of the reads that were from 50 to 1000 nucleotides in length. In contrast to the negative control, several reads from the synthetic ssDNA sample did not show any similarity with the sequences in the non-redundant nucleotide GenBank database, and showed stretches of DNA lacking nucleobase cytosine (SEQ ID NO:6-8, the nucleotides that correspond to the segments of the injected order of the nucleotides are specially underlined with one or two solid lines or with a dashed line; a long stretch of one nucleotide should be read as a single nucleotide):

[0140] SEQ ID NO: 6 a a a a tggggg gtttaggggt taaagggggg ggtttttttt tttttttttt tttttttttt tttttttttt tataaaaa

[0141] SEQ ID NO: 7 aggatggata gagtaaatag agtagattgt aatataaggg aaaggttaaa gagagggtgt ttttgttttt tttttttttt ttgaaaaatt ttttttttgg gggaaaaaaa aaaaaaaaaa

[0142] SEQ ID NO: 8 tttaggataa gagaatggtg gaaggagagt agagaagggt gagagtggag attttttttt tttttttttt ttttttgggg aaaaaaaaaa aaaaaaaaa_a_ aaaaaaaaaa _aaaa_aaaaaa aaaaaaaatt _ttttttttt_ _gaaatgtgag atgtagggaa tgtgaagagg agaaaaaaga ttgaaatagt gaagaagttt ggtgaggggg aagtaaaaaa aaaaaaaaaa aat

[0143] Example 5: Synthesis of ssDNA, release by Proteinase K and removal of contaminating DNA by restriction enzymes.

[0144] To further support that the present approach enables the synthesis of ssDNA with specific DNA sequence using AbiK and normal nucleotides, we: (i) immobilized AbiK on four flow cells of the SPR chip CM5; (ii) injected nucleotides dATP or dCTP or dTTP or dGTP (all at concentration of 100 pM ) at flow rate 20 pl / min and 25°C for selected injection time (60 - 200 s) across the chip-immobilized AbiK (it is of note, that we injected the dGTP, and dCTP to govern the annealing of Cig DNA primer used for PCR amplification of the synthetic DNA); (iii) ejected the SPR chip carrying the synthetic ssDNA covalently attached to AbiK from the SPR apparatus and we treated the surface of the chip by proteinase K (0.4 mg / mL in Equi Phi29™ DNA Polymerase buffer at 55°C for lh); (iv) afterwards, proteinase K was inactivated at 95°C for 15 min; (v) reaction mixture was cooled down and the fastdigest restriction enzymes Dpnl (target DNA sequence 5'- GATC-3'), Bspl43l (target DNA sequence 5'-GATC-3') and Alul (target DNA sequence 5'-AGCT-3'), all Thermo Fisher Scientific), were added at 0.2 U into the reaction mixture to degrade the DNA contaminations carrying nucleotides containing cytosine (37°C, 30 min); next (vi) the ssDNA polymerised by AbiK was amplified by PCR using Cig DNA primer and Phusion DNA polymerase (both Thermo Fisher Scientific; according to manufacturer's instructions with annealing temperature of 55°C and 2 min of elongation time at 72°C for 35 cycles); and (vii) sequenced using Oxford Nanopore Technology, Full PlasmidSeq service provided by company Microsynth. As a negative control, in the control reaction mixture the water was used instead of the sample containing the synthesized DNA and the reaction was carried out as described in steps iii-viii.

[0145] Results in FIGURE 10 show the polymerisation of DNA composed of nucleotides carrying either nucleobase cytosine, adenine, thymine, or guanine. We injected the nucleotides in the order (C - dCTP, G - dGTP, A - dATP, T - dTTP) to obtain SEQ ID NO: 9:

[0146] 5 -C-C-A-T-A-T-A-T-A-T-A-T-A-T-A-T-A-T-A-T-A-T-G-G -3'.

[0147] By using nanopore sequencing we obtained 14 sequencing reads and a sequencing read for the control sample. In contrast to the negative control, almost half of the reads from the synthetic ssDNA sample did not show any similarity with the sequences in the non-redundant nucleotide GenBank database (SEQ ID NO:10-15; the nucleotides that match the segments of the injected order of the nucleotides are specially underlined with one or two solid lines or with a dashed line; a long stretch of one nucleotide should be read as a single nucleotide):

[0148] SEQ ID NO: 10

[0149] SEQ ID NO: 11 CCCATCCAAA AAAAAAAAAA AAAAAATTTT TTTTAAAAAA AAAAAAAAAA TTTTTTTTTT AAAAAAAAAA AATT I I I I I I I I I I I I TTAA AAAAAATTTT TT

[0150] SEQ ID NO: 12

[0151] AMAMAMA^AMMM_AA AAA_AAAAAAT J J JJ111LLL HULL LLL TGGGGGGC

[0152] SEQ ID NO: 13

[0153] AAAAAAAAAA AAAAAAAAAA AATTTAAAAA AAATTTTAAA AATTT

[0154] SEQ ID NO: 14

[0155] TTTTAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

[0156] AAAAAAAAAA AAAAAAATTT TTTTTAGATT TTT I I I I I I T TTAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAATTTTTT T I I I I I I I TT TAAAAAAAAA AAAAAATTAA AAAAAAAATT TTTTTTTTTT TTTTTTT

[0157] SEQ ID NO: 15

[0158] AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA TTTTTTTTAA AAAAAAAAAA ATAAAATTTT TTT A

[0159] Example 6: Regeneration of solid surface for renewed synthesis.

[0160] To regenerate the solid surface, AbiK can be (instead of covalent attachment to the solid surface e.g. via amino-coupling to the CM5 SPR chip) immobilized to the solid surface covered with (C)zo oligonucleotide composed of 20 cytosine nucleobases (FIGURE 11). Prior to injection, AbiK is incubated with 100 pM dGTP in the buffer containing 20 mM Tris pH 8.3, 140 mM NaCI, 2mM MgCL, 0.005% P20, to generate a short stretch of ssDNA rich in guanines that hybridize to the (C)zo oligonucleotide. This can be reversed by 10-50 mM NaOH to regenerate the (C)zo oligonucleotide surface.

[0161] Example 7: Encoding information in DNA using bacteriophage abortive infection system reverse transcriptase AbiK. To demonstrate that information can be stored in DNA synthesized by AbiK, we designed an assembly to synthesize ssDNA encoding the word "DNA" and read the stored information (FIGURE 12A). Notably, the AbiK was prompted to synthesize a sequence encoding the word "DNA" twice, flanked by a hompolymeric guanine nucleobase sequence, which enabled data retrieval. The sensorgram indicates that AbiK on the chip incorporated the deoxynucleotides with each injection, suggesting that the information was encoded in the DNA (FIGURE 12B). However, on the basis of the sensorgrams alone, we cannot determine whether the activity of the AbiK enzyme molecules immobilized on the chip was homogeneous, that is, whether all enzyme molecules came into contact with nucleotides and used the incoming nucleotides to extend the DNA strand. Therefore, we released the synthetic ssDNA from the SPR chip by AbiK proteolysis using proteinase K and generated the second strand using the large (Klenow) fragment of E. coli DNA polymerase I and an oligo-Cig primer (FIGURE 12A). For this we removed the SPR chip containing the synthetic ssDNA covalently bound to AbiK from the SPR apparatus and treated the surface of the chip with proteinase K (0.4 mg / mL in 50 pL EquiPhi29™ DNA polymerase buffer) at 55°C for lh. After scoring the chip surface with a pipette tip, we transferred the mixture to a microcentrifuge tube and reconstituted the evaporated water to 50 pL. Proteinase K was then inactivated by incubation at 95°C for 15 minutes, after which the mixture was cooled on ice for 5 minutes. To synthesize the second strand, we added 0.8 pM 18-mer poly-C oligonucleotide, 0.2 mM dNTPs and Klenow fragment reaction buffer at working concentration. We then heated the suspension to 95°C for 2 minutes and cooled it on ice for 5 minutes. We then added the Klenow fragment lacking both the 3'^5' and 5'^3' exonuclease activity of DNA polymerase I (Thermo Fisher Scientific, USA) at a concentration of 0.25 U / pl and incubated the mixture at 25° C for 15 minutes and then at 37° C for 1 hour. The Klenow fragment was then thermally inactivated by incubation at 75°C for 15 minutes and samples sent for sequencing. Nanopore sequencing revealed that the DNA produced by AbiK consisted of synthetic fragments of "'150-1500 bp. In the DNA fragments, multiple ~10- 400-bp-long stretches of DNA consisting of only three of the four nucleotides were detected (SEQ ID NO: 16-19). Moreover, DNA regions in which the segments signified the injection patterns (FIGURE 2A) and represented specific letters (Table 2) or even the whole word "DNA" (SEQ ID NO: 16) were observed. The different sections of DNA are marked according to the nucleotide triplet: ATC, TCG, ACG, and ATG.

[0162] SEQ ID NO:19-22; The nucleotides that match the segments of the injected order of the nucleotides are specially underlined with one or two solid lines, wavy line or with a dashed line; a stretch containing only three out of four nucleotides should be read as the absent nucleotide. In one of the sequences is shaded in grey the homopolymeric guanine nucleobase sequence, which flanked the word "DNA". (Note that certain sequences carry a single non underlined nucleotide, which is presumably a sequencing error.):

[0163] SEQ ID NO: 19

[0164] AATATCAAAAAAATATCCAAACAAAATATACTCACACATATAAACCTGTGTGTTGCGTGGAAAG.AGAAAGA

[0165] AGAAAGAGCGCGCGCGCTGTGCGTCCCTCGCTGTGTCACAAAATACCCAATACTCAAATAATAAACAAAAT

[0166] CAATAAAATAAAAAGGAAGAACGAAAGAGCGAAGAGAGAGAAAGA ^

[0167] ACACCCAAAATTAATACCCATATATCAAACCATATATAAACACAACATAAAGAGTGAGAGAGTTTGAAGGA G GG GGAAGGATGGATAAGAATGGGAGAGAGAAATGAATAGGTAGA ^

[0168] TA AAGAAG AGAATAAAGATGAAGTGA GHGAGAAGAGAGA AGAATAATAAGAGAGTAAAAAGGAGCGCGGCTAG

[0169] SEQ ID NO: 20

[0170] CATCAATAAAAACACAAAAACACCCAACAATAAAAAAAAAATATAAATAAAAACAAAAATATCAAAAAAAC CTTTACATAAATAAAAAACTATTAAAACCTTAAAACTCATACAAATTCGCGTCGCTGCTGTCCGGCGTCGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGACATTTTACATACC AAACATTATCCAACCTACCATTATTCAACTACCAATCCACCAAATACCAACCTTAATCACAAACCAATAAAAA CTACCACAGGTTCGTGGCGTCCGCGCGTGCTCCGCGGGCAGTTGTCTGTTCGCCGCTCGTGCCGACGCCAC GAGACCT ATAGTAGTTTTATTGGTTTT AT AATTGTGGTATTTGTCGTAGAATTGGTCATTGATAAGATGGTT AAGTTGGATATGTTTTAATGT

[0171] SEQ ID NO: 21

[0172] AATAAAAATACCAAAAACATAATAACTCACAAAAAAAACAAAAAAAAAACAACTATTAATAAAACAACCCA

[0173] AACCATCATAAAACAAGCAGAGAAACGCAGCAGGGTTCTCCAAAATAAAACCCCAAATTTAGAACATACAC ACTCCAATCAAAACACCAAAATTCAAACACATAAAACATCCACCCCTTCATTCAAACCTATAATAATCTATTA ACATTCACACATACACAATCATTTTACAATATATACATAAATACAACACAACTAAATACAACTTATATTACAA CTACCAACACACACAAAGCCCAAACCAACACATTATTAACTAATTCTAAACAAAAAATATACCAACCCTAAA ATAAAACAAAAACAAAAACTTAAATTAACCAATCCAAATCAATTTAATATAACTATAATCACATCCACCTCTT AAATACAATAAACATTTAACACACACCAACTCTCCATCTCTAAATCATACACTAATTAATTATACCAATAATA

[0174] ATTCCACTAAACATGGTACCTGGGCTGCCGGGCGCGGCTTGTGCGTCGTGACGAAGGGGCAAGGGAGGG AGCGGGCGGTGTGGGGGCGCCCGTTCGCTGTGTGCCCCGCCGGTGGCTGCTAAACAAAATAATAAAAATA AAAATATTCAAAATCCAAAAAATTAATATTTACACATAACCATATGAACTAAAACTCTACCCTCATCAAAACA AAACATAAAAAAAAATACAAAAACCCTACCATATACAAAAATTCCAAATAAATCATAAAAAATAATATTAAC CAAACTATCATACACAACCAAATAACACCTCCTATTAAAAAAAAACCAATAACAATAAACACCTCCGAAAAG AGAGAAGCGGAACCTTTACCCCTCAACATAACCAAAAACAAAATCCCCTATAGTATGATATCTAAAACCTCC TTTATAAACAAAAAAAAAAATCTTAACCCACAAAAAACTATACATATAAATCCATCATACCAATAAAAACTC TCAACTTCTACAAACAAATAAAAATTACAAATCAAAATAAAAAACACCACAACCCAAATTACCATAATAAAC

[0175] AACACGCCGCACAAGCCGCGCCCCGGGCAGCCCAGAGAAATTTATGTTAGTGGAATTAATTTATTGGTATA GTTTAATTAGTGTATGATTTAGAGATGGGTATTAGTTGGTGTGTGTTAAATGTTATTGTATTTTAGAGGTGG ATGTGTGATTAATAGTTATATTAAATTGATTTGGATTGGTTAATTTAAAGTTTTTGTTTTTGTTTTATTTTAGG

[0176] GTAGATCTTTTTGTTTAGAATTAGTTAATGTGTTGGTTTGGGGTTTTTGGGTGTGTTGGTAGTTGTAATATA

[0177] AGTTGTATTTCGTTGTGTTGTATGTATATATTGTAAAATGATTGTGTATGTGTGAATGTTAATAGATTATTAT

[0178] CGTAAATGAAGGGGTGGATGTTTTATGTGTTTGAATTTTGGTGTTTTGATTTTGGGAGAGTGTGTAATGTT

[0179] GAAATTTGGGGTTTATTTTGGAGAACCCTGCTGCGTTTCTCTGCTTGTTTTATGATGGTTTGGGTTGTTTATT

[0180] AATAGTTGT I I I I I I GT I I I I I I I TGCCTAAGTTAGTTATCTTTTGGTGAT

[0181] SEQ ID NO: 22

[0182] CTTGCCGGGGTTTGGGGCGTCCGCGGGGGTGGCGGGGCCGGGGCGGTTGGCGTGCCGGGCGCGTCCGT

[0183] CGTCCGCGCGCGCGTCTTGCCGCGGTTTTGGTCGTTGGGCGTGGGGCGCTCGTGCGCGTGTCGAAGCAGG

[0184] GACGAGAGCGCGACAAGAGAAGAGAAAACCAAAAGAGACGAAGAGAAAGGAGCGGGGAAAGACGGGC

[0185] GCAAAAAGGAGAGAGAGCAAGAAGGGAGAAAGGAGAAAACACACAAAAAAAGAACCGGCAGAGGGGA

[0186] AAGAACGAGAACGAACCGCACACGGAAAGCGCACAAAATATAAACAAAAAATTCTAACACTAACTATAAA

[0187] ACACAACAACTCTCACTACACCTTAAAACCCACTAAAACAAATAAAAAATACAATAAACACTTAATCCAAAA

[0188] AAAAAACTACCACATAAAACATTAAAAAATATACTTCTATATTAATTAAAAAACAACAACTAAAAAAAACGA

[0189] AGAGAAAG CGGGACAAA ACCAACAAAATATAAACCTTATTCAACCCAAATCGGGTTAAAAGGTTAT

[0190] GGAGTATAATAAGATACGTCGGTGTGGGTGGTGGTCCGGGGCGTGGCGCTGGCCGCCGCGCCGGACCCA

[0191] CCGCCCACCGACATATCTTATTATACTCCATAACCTTTTAACCCGATTTGGTTGAATAAGGTTTATATTTTGTT

[0192] GGTTTTTTGTCCCGTTTCTCTTCGTTTTTTTCAGGTACTTCTTTTAATTAATATAGAAGTATATTTTTTAATGTG

[0193] ATGTGGTAGTTTTTTGAGATAAAATATTTTATTGTATTTTTTATTTGTTTAGTGGGGTAAGAGTGTAGTGAG

[0194] AGTTGTTGTGTTTTATAGTTAGTGTTAGAATTTTTTGTTTATATTTTGTGCGCTTTCCGTGTGGGTCCGTTCTC

[0195] GTTCTGCCGGTTCTTTTTTTGTGTGTTTTCTCCTTTCTCCTCTTCCTCCTCCTTCTTTTTTGCGCCCGTCTTTCCC

[0196] CGCTCCTTTCTCTTCGTCTCTTTTGGTTTTCTCTTCTTCCTCTGTCGCGCTCTCGTCCTGCTTCGACACGCGCAC

[0197] GAGCGCCCGCAACAAACCAAAACCGCGGCA

[0198] Table 2. The three-nucleotide code used to encode information using the bacteriophage abortive infection system reverse transcriptase AbiK (bases from 5'- to -3': sequences of nucleotides carrying one of the adenosine, thymidine, cytosine or guanine base).

[0199] Number assigned to the Three-nucleotide mix combination

[0200] ATC 1

[0201] ATG 2

[0202] TCG 3

[0203] ACG 4

[0204] Numerical

[0205] Letter of the alphabet code

[0206] A 123

[0207] B 124 c 132

[0208] D 134

[0209] E 142

[0210] F 143

[0211] G 213

[0212] H 214

[0213] I 231

[0214] J 234

[0215] K 241

[0216] L 243

[0217] M 312

[0218] N 314

[0219] O 321

[0220] P 324

[0221] Q 341

[0222] R 342

[0223] S 412

[0224] T 413

[0225] U 421

[0226] V 423 w 431

[0227] X 432

[0228] Y (Q+Q) 341341

[0229] Z (X+X) 432432 space dNTP mix

[0230] Example 8: Showing that enzyme AbiA immobilized on the solid support retains activity and allows for user-directed nuleotide incorportation.

[0231] Here we use purified recombinant AbiA (10) and have immobilized it on the SPR chip to show that the protein remains functional and can incooroporate selected dNTPs in the growing DNA strand.

[0232] To do this, we used the injection system of the SPR apparatus to inject the selected dNTP over AbiA as a substrate, which incorporates dNTPs into the growing ssDNA chain covalently attached to the AbiA (FIGURE 13). AbiA at concentration 1.5 mg / ml was dissolved in sodium acetate buffer (pH 5.6) to the final concentration of 50 pg / ml. The 50 pg / ml of AbiK diluted in 10 mM Na-acetate, pH 5.0, was applied on the flow cell 2 and 50 pg / ml of AbiA diluted in 10 mM Na-acetate, pH 5.6, to the flow cell 4. We followed the immobilisation procedure as it is described in Example 1. In such a way "'6500 RU of AbiK or 4700 RU of AbiA were immobilized to the flow cell 2 or to the flow cell 4, respectively. The first and third flow cell was left unmodified and served as a control for non-specific binding of tested compounds. Either individual dNTPs (at "'100 pM) or a mixture containing dATP, dCTP, dTTP, dGTP (each at ~100 pM), or 100 pM dUTP, was injected in the running buffer composed of 20 mM Tris pH 8.3, 140 mM NaCI, 2 mM MgCL, 0,005% P20, separately, over the chip- immobilized AbiK or AbiA. In addition, dNTPs were also injected in a running buffer containing manganese instead of magnesium. This buffer was composed of 20 mM Tris pH 8.3, 140 mM NaCI, 2 mM MnCL, 0,005% P20, over the chip-immobilized AbiK or AbiA. Results show that AbiA immobilized to the solid surface is functional and can incorporate dNTPs into the growing DNA strand in a user-controlled manner (FIGURE 13, FIGURE 14). When experiments were performed in the running buffer containing MgCL, AbiA incorporated in 120 second injections ~ 32, 20, 2, 78, or 13 response units of dCTP, dTTP, dGTP, dATP, or dUTP, respectively (FIGURE 14).

[0233] Amino acid sequence of recombinant AbiK.

[0234] SEQ ID NO. 1: (600 aa)

[0235] GSKKEFTELYDFIFDPIFLVRYGYYDRSIKNKKMNTAKVELDNEYGKSDSFYFKVFNM ESFADYLRSHDLKTHFNG KKPLSTDPVYFNIPKNIEARRQYKMPNLYSYMALNYYICDNKKEFIEVFIDNKFSTSKFFNQLNFDYPKTQEITQTL LYGGIKKLHLDLSNFYHTLYTHSIPWMIDGKSASKQNRKKGFSNTLDTLITACQYDETHGIPTGNLLSRIITELYMC HFDKQMEYKKFVYSRYVDDFIFPFTFENEKQEFLNEFNUCRENNUINDNKTKVDNFPFVDKSSKSDIFSFFENITS TNSNDKWIKEISNFIDYCVNEEHLGNKGAIKCIFPVITNTLKQKKVDTKNIDNIFSKRNMVTNFNVFEKILDLSLKD SRLTNKFLTFFENINEFGFSSLSASNIVKKYFSNNSKGLKEKIDHYRKNNFNQELYQILLYMVVFEIDDLLNQEELLN LIDLNIDDYSLILGTILYLKNSSYKLEKLLKKIDQLFINTHANYDVKTSRMAEKLWLFRYFFYFLNCKNIFSQKEINSYC QSQNYNSGQNGYQTELNWNYIKGQGKDLRANNFFNELIVKEVWLISCGENEDFKYLN

[0236] Nucleotide sequence of recombinant AbiK.

[0237] SEQ ID NO. 2: (1800 nt) ggatccaaaaaagagtttactgaattatatgattttatatttgatcctatttttcttgtaagatacggctattatgatagatctattaaaaacaa aaaaatgaatactgcaaaagttgaattagacaatgaatatggaaaatcagattctttttattttaaagtatttaatatggaatcctttgcagat tatttaaggagtcatgatttaaaaacacattttaacggtaaaaaacctctatcaacagacccagtatattttaatattccaaaaaatatagaa gctagaagacaatataagatgcccaatttatacagttatatggcattaaattattatatatgtgacaataaaaaagagtttatagaagtattt attgataacaaattttcaacgtcaaaattttttaatcaattgaattttgattatcctaagacacaagaaattacacaaacattattatatggag gaataaagaaattacatttagatttatctaatttttatcatactttatatacacatagtataccatggatgattgatggaaaatctgcatctaaa caaaatagaaaaaaagggttttctaatacattagatactttgattacagcttgtcaatacgacgaaacacatggcattccaactggaaatcta ttgtctaggattattaccgaactatatatgtgccattttgataaacaaatggaatataagaagtttgtgtattcaagatatgtagatgattttat atttccgtttacttttgagaatgaaaagcaagaatttttaaatgaatttaatctaatctgtcgagaaaataacttaattattaatgataataaaa cgaaagttgacaatttcccgtttgttgataaatcgagtaaatcggatattttttctttttttgaaaatattacttcaactaattccaacgacaagt ggattaaagaaataagcaattttatagattattgtgtgaatgaagaacatttagggaataagggagctataaaatgtattttcccagttataa caaatacattgaaacaaaaaaaagtagatactaaaaatatagacaatatcttttcgaaaagaaacatggttaccaattttaatgttttcgaa aaaatattagatttatcattaaaagattcaagattaactaataagtttttgactttctttgaaaatattaatgaatttggattttcaagtttatca gcttcaaatattgtaaaaaaatattttagtaataattcaaagggcttaaaagaaaaaatagaccactatcgtaaaaataattttaatcaaga attatatcaaatattgttgtatatggttgtctttgaaatagatgatttattaaatcaagaagaattactaaacttaattgatttaaatattgatga ttattctttaattttagggacgattttatacctaaagaatagttcatataaattggaaaaattattaaaaaaaatagatcaattatttattaata ctcatgccaactacgacgttaaaacttctcgtatggcagaaaaattatggctatttcgttatttcttttattttttaaattgtaagaatatttttag tcaaaaagagataaatagttattgtcaatctcaaaactataattcaggacagaacggatatcaaacagaacttaattggaattatattaaag gtcaagggaaggatcttagagcgaataacttttttaatgaattgatagtaaaagaagtttggttaatttcttgtggtgagaacgaagatttca aatatttaaat

[0238] Nucleotide sequence of AbiK, NC_004960.1x5096-3297 Lactococcus lactis plasmid pSRQ800

[0239] SEQ ID NO. 3: (1800 nt) atgaaaaaagagtttactgaattatatgattttatatttgatcctatttttcttgtaagatacggctattatgatagatctattaaaaacaaaaa aatgaatactgcaaaagttgaattagacaatgaatatggaaaatcagattctttttattttaaagtatttaatatggaatcctttgcagattatt taaggagtcatgatttaaaaacacattttaacggtaaaaaacctctatcaacagacccagtatattttaatattccaaaaaatatagaagcta gaagacaatataagatgcccaatttatacagttatatggcattaaattattatatatgtgacaataaaaaagagtttatagaagtatttattga taacaaattttcaacgtcaaaattttttaatcaattgaattttgattatcctaagacacaagaaattacacaaacattattatatggaggaata aagaaattacatttagatttatctaatttttatcatactttatatacacatagtataccatggatgattgatggaaaatctgcatctaaacaaaa tagaaaaaaagggttttctaatacattagatactttgattacagcttgtcaatacgacgaaacacatggcattccaactggaaatctattgtct aggattattaccgaactatatatgtgccattttgataaacaaatggaatataagaagtttgtgtattcaagatatgtagatgattttatatttcc gtttacttttgagaatgaaaagcaagaatttttaaatgaatttaatctaatctgtcgagaaaataacttaattattaatgataataaaacgaaa gttgacaatttcccgtttgttgataaatcgagtaaatcggatattttttctttttttgaaaatattacttcaactaattccaacgacaagtggatta aagaaataagcaattttatagattattgtgtgaatgaagaacatttagggaataagggagctataaaatgtattttcccagttataacaaata cattgaaacaaaaaaaagtagatactaaaaatatagacaatatcttttcgaaaagaaacatggttaccaattttaatgttttcgaaaaaata ttagatttatcattaaaagattcaagattaactaataagtttttgactttctttgaaaatattaatgaatttggattttcaagtttatcagcttcaa atattgtaaaaaaatattttagtaataattcaaagggcttaaaagaaaaaatagaccactatcgtaaaaataattttaatcaagaattatatc aaatattgttgtatatggttgtctttgaaatagatgatttattaaatcaagaagaattactaaacttaattgatttaaatattgatgattattcttt aattttagggacgattttatacctaaagaatagttcatataaattggaaaaattattaaaaaaaatagatcaattatttattaatactcatgcc aactacgacgttaaaacttctcgtatggcagaaaaattatggctatttcgttatttcttttattttttaaattgtaagaatatttttagtcaaaaa gagataaatagttattgtcaatctcaaaactataattcaggacagaacggatatcaaacagaacttaattggaattatattaaaggtcaagg gaaggatcttagagcgaataacttttttaatgaattgatagtaaaagaagtttggttaatttcttgtggtgagaacgaagatttcaaatattta aattga

[0240] SEQ. ID NO: 4 AbiA from Lactococcus lactis

[0241] MITLQHQDWERAVNMIKNIPPSAKNKYFQTFPFFLLSETSWEELLSENFFYSYIKSGEFLTYQENLSFYDRTIQKS

[0242] HGAYRQTRIVSPIIYIFLIAIASQVERIYVEKRTNDMSVYFSGSFEKEKNTAHYKQSYNTYMTELNACQEEFDYYFQ

[0243] TDFSTFFHLVDTDNLFNKIDRLDPKSALVYSSLIKMIGQGRM PIVDGNSGLSFLNTVVYLDDFDKEIIDSLKTIVEIE

[0244] SFKLVRYVDDLHIFIKCANKDLDFLNYKVYNLLCEKATKHHLEINSSKTKSFTPTSELSTKMNTDLYNFFVYNEDVD

[0245] FEQYFSKNTLIEFLDKLNNMSVNADFSEYEKEVLYTLENPEIVSDGSYILNAIVYNKSTWSQDYDIKNKISLLVNSN

[0246] YRKLRYSAKALITLVLNTRDGDIIKGLLNNLFTTFKNGTNDIIDEIILIEYLVQRKFNHKDLMTILKADDHGIKEYIKAY

[0247] QTSDFIKSLEKNKVIFYTNQKEVYPLISKDKILNFIYFRAKYFESLDLVLESFAYYKNYFDRFVAHAMFCTGIDSGRK PNYKLYYTEGKLIDGLKQLNFLSSDEITKIINEAHKIRNSNPVSHSSAGLLQNEDFSRYRVKSSLNDLKIIIEQLSTLLQ NKNRL

[0248] References.

[0249] 1. Hoose A, Vellacott R, Storch M, Freemont PS, Ryadnov MG. 2023. DNA synthesis technologies to close the gene writing gap. Nat Rev Chem 7:144-161.

[0250] 2. Jensen MA, Davis RW. 2018. Template-Independent Enzymatic Oligonucleotide Synthesis (TiEOS): Its History, Prospects, and Challenges. Biochemistry 57:1821-1832.

[0251] 3. Yu M, Tang X, Li Z, Wang W, Wang S, Li M, Yu Q, Xie S, Zuo X, Chen C. 2024. High- throughput DNA synthesis for data storage. Chem Soc Rev.

[0252] 4. Palluk S, Arlow DH, De Rond T, Barthel S, Kang JS, Bector R, Baghdassarian HM, Truong AN, Kim PW, Singh AK, Hillson NJ, Keasling JD. 2018. De novo DNA synthesis using polymerasenucleotide conjugates. Nat Biotechnol 36:645-650.

[0253] 5. Lee HH, Kalhor R, Goela N, Bolot J, Church GM. 2019. Terminator-free templateindependent enzymatic DNA synthesis for digital information storage. Nat Commun 10. Fortier LC, Bouchard JD, Moineau S. 2005. Expression and Site-Directed Mutagenesis of the Lactococcal Abortive Phage Infection Protein AbiK. J Bacteriol 187:3721-3730. Emond E, Holler BJ, Boucher I, Vandenbergh PA, Vedamuthu ER, Kondo JK, Moineau S. 1997. Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism AbiK from Lactococcus lactis. Appl Environ Microbiol 63:1274-1283. Wang C, Villion M, Semper C, Coros C, Moineau S, Zimmerly S. 2011. A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro. Nucleic Acids Res 39:7620-7629. Figiel M, Gapinska M, Czarnocki-Cieciura M, Zajko W, Sroka M, Skowronek K, Nowotny M. 2022. Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases. Nucleic Acids Res 50:10026-10040. Gapinska M, Zajko W, Skowronek K, Figiel M, Krawczyk PS, Egorov AA, Dziembowski A, Johansson MJO, Nowotny M. 2024. Structure-functional characterization of Lactococcus AbiA phage defense system . Nucleic Acids Res 52:4723-4738.

Claims

Claims1. Method for making one or more oligo- or polynucleotides comprising the steps:(a) Optionally combining a nucleotide triphosphate(s), one or more cations, and an abortive infection system reverse transcriptase (Abi) enzyme which is immobilized at a solid surface, to facilitate a reaction to attach the nucleotide covalently to the Abi enzyme, such as to form a nucleotide-Abi enzyme conjugate,(b) Combining a nucleotide triphosphate(s), one or more cations, and an Abi enzyme- nucleotide conjugate which is immobilized at a solid surface, to facilitate a reaction to add the nucleotide into a growing ssDNA covalently attached to the Abi enzyme,(c) Repeating step (b) until the one or more oligo- or polynucleotides is / are formed;(d) Applying means to release the one or more oligo- or polynucleotides off said solid surface; such as to obtain said one or more oligo- or polynucleotides.

2. The method according to claim 1, wherein step(a) is performed once and step (b) is performed at least two times.

3. The method according to claim 1, wherein step (a) is not performed.

4. The method according to any one of the preceding claims, wherein said Abi enzyme is AbiK from Lactococcus lactis or a functional derivative thereof, such as a protein carrying an Abi-type, functional reverse transcriptase domain, exemplified by the AbiK reverse transcriptase domain (PROSITE entry PS50878), encompassing residues 1 to 326 of SEQ ID NO:1.

5. The method according to any one of the preceding claims wherein said Abi enzyme is a polypeptide having at least 80% sequence identity to SEQ ID NO:1, at least 85% sequence identity to SEQ ID NO:1, at least 90% sequence identity to SEQ ID NO:1, at least 95% sequence identity to SEQ ID NO:1, at least 98% sequence identity to SEQ ID NO:1.

6. The method according to any one of the preceding claims, wherein said AbiK protein from Lactococcus lactis or a functional derivative thereof is a protein having at least 85% identity to SEQ ID NO: 1.

7. The method according to any one of the preceding claims, wherein said AbiK protein from Lactococcus lactis or a functional derivative thereof is a protein having at least 95% identity to SEQ ID NO: 1.

8. The method according to any one of the preceding claims, wherein said AbiK protein from Lactococcus lactis or a functional derivative thereof is a truncated derivative of SEQ ID NO:1 exhibiting at least 85% identity or at least 90% identity to the corresponding part of SEQ ID NO:1.

9. The method according to any one of the preceding claims, wherein said Abi enzyme is AbiK from Lactococcus lactis, SEQ ID NO: 1.

10. The method according to any one of the preceding claims, wherein said Abi enzyme is AbiK from Lactococcus lactis or AbiA from Lactococcus lactis, or a protein exhibiting sequence homology to AbiK or AbiA.

11. The method according to any one of claims 1-3, wherein said Abi enzyme is AbiA from Lactococcus lactis or a functional derivative thereof.

12. The method according to any one of the preceding claims, wherein said solid surface is a surface plasmon resonance (SPR) sensor chip.

13. The method according to claim 12, wherein the repetitions of step (b) is followed using by a surface plasmon resonance refractometer.

14. The method according to claim 12 or 13, wherein the timing of the repetitions of step (b) is controlled by a surface plasmon resonance refractometer.

15. The method according to any one of the preceding claims, wherein said nucleotide triphosphate is injected across the Abi enzyme to facilitate a reaction to add the nucleotide into a growing ssDNA covalently attached to the Abi enzyme.

16. The method according to claim 15, wherein said injection of nucleotide triphosphate is performed using a microfluidic system.

17. The method according to claim 15 or 16, wherein the reaction to add the nucleotide is being followed in real time in an automated set-up.

18. The method according to any of the preceding claims, wherein in steps (a) and (b) said one or more cations comprises Mn2* and / or Mg2+.

19. The method according to any of the preceding claims, wherein in steps (a) and (b) said nucleotide triphosphate, one or more cations, and an Abi enzyme or a functional derivative thereof are combined in a buffer comprising a surfactant such as polysorbate 20.

20. The method according to any of the preceding claims, wherein in steps (a) and (b) said nucleotide triphosphate, one or more cations, and an Abi enzyme are combined in a buffer comprising Tris, NaCI, MgCL, and a surfactant.

21. The method according to any of the preceding claims, wherein in step (a) said nucleotide triphosphate, one or more cations, and an Abi enzyme are combined in a buffer comprising 20 mM Tris pH 8.3, 140 mM NaCI, 2 mM MgCL, 0.005% polysorbate 20.

22. The method according to any one of the preceding claims, wherein in step (c) said means to release the one or more oligo- or polynucleotides off said solid surface is selected from one or more from the group consisting of a suitable restriction enzyme, an oligonucleotide, a protease and a reagent, such as 20 mM Tris-HCI (pH 8.8 at 25°C), 10 mM (NH4)2SO4, 10 mM KCI, 0,1 mg / mL BSA, 1% (v / v) Triton X-100, 2 mM MgSO4.

23. The method according to any one of the preceding claims, wherein said means to release the one or more oligo- or polynucleotides is proteinase K.

24. The method according to any one of the preceding claims, wherein said means to release the one or more oligo- or polynucleotides is USER enzyme (Uracil-specific excision reagent (USER) comprising an enzyme mix of uracil DNA glycosylase and DNA glycosylase-lyase).

25. The method according to any one of the preceding claims, further comprising a step wherein the released oligo- or polynucleotide ssDNA in treated with one or more restriction enzymes to remove contaminating DNA.

26. The method according to any one of the preceding claims, wherein said nucleotide triphosphate is selected from the group consisting of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxyuridine triphosphate (dUTP).U. The method according to any one of the preceding claims, wherein said oligo- or polynucleotide contains from 5 to 200 nucleobases, from 5 to 100 nucleobases, from 5 to 50 nucleobases, from 5 to 30 nucleobases, from 20 to 200 nucleobases, from 20 to 100 nucleobases, from 20 to 50 nucleobases, or from 5 to several hundred nucleobases.

28. Composition comprising a collection of several oligo- or polynucleotides which all encode or display the alternating segments of nucleotides when a stretch of the alternating segments containing only three of the four nucleotides and the absent nucleotide in a segment is considered as encoding or displaying the information.

29. The method according to any one of the claims, wherein said Abi enzyme is AbiA from Lactococcus lactis, SEQ. ID NO: 4.

30. Composition comprising a solid surface whereupon an Abi enzyme is immobilized in at least one location.

31. The composition according to claim 30, wherein said Abi enzyme is AbiK from Lactococcus lactis or a functional derivative thereof.

32. The composition according to claim 30, wherein said Abi enzyme is AbiA from Lactococcus lactis or a functional derivative thereof.

33. The composition according to any one of claims 30-32, wherein said solid surface is a surface plasmon resonance (SPR) sensor chip.

34. The composition according to any one of claims 30-33, wherein said Abi enzyme is immobilized to said solid surface by amine-coupling.

35. The composition according to any one of the claims 30-34, wherein said immobilization is by (i) surface activation by l-ethyl-3-(3-dimethylpropyl)-carbodiimide (EDC) and N- hydroxysuccinimide (NHS), (ii) coupling of the Abi enzyme, and (iii) surface blocking by ethanol amine.

36. The composition according to any one of claims 30-33, wherein said solid surface is covered by oligonucleotides comprising from 10-30 identical nucleotides, and said Abi enzyme has been incubated with the nucleotide triphosphate of the matching nucleotides, such that immobilizationis by way of hybridization between the oligonucleotides on said solid surface and the oligonucleotide formed by the Abi enzyme and attached thereto.

37. The composition according to claim 36, wherein said solid surface is covered by (C)i5-2s (oligonucleotide having from 15 to 25 cytosines) and the Abi enzyme is incubated with dGTP.

38. Method for regenerating the composition as defined in any of claims 30-35, comprising contacting of the at least one location of said composition with a solution comprising a nuclease, such as benzonase.

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