Nucleic acid polymerases and their use in producing non-DNA nucleotide polymers - Patents.com

Abstract

In one aspect, the present invention relates to nucleic acid polymerases capable of producing non-DNA polymers. Additionally, the present invention relates to uses of said polymerases and the resulting products.

Description

[Technical field]

[0001] FIELD OF THEINVENTION In one aspect, the present invention relates to nucleic acid polymerases capable of producing non-DNA polymers. Additionally, the present invention relates to uses of said polymerases and the resulting products. [Background technology]

[0002] BACKGROUND OF THEINVENTION Chemical alterations to standard (deoxy)ribonucleic acids are of great interest in the overlapping fields of medicinal chemistry and nucleic acid-based therapeutics (including RNA vaccines), as well as in the synthetic and chemical biology of nucleic acids. These modifications include a wide range of isomeric substitutions, sugar changes, sugar substituent modifications, and nucleobase modifications, including but not limited to altered glycosidic linkages, unnatural base pairing interactions, and modified backbone chemistries. Of these, modifications to the 2'-hydroxy group of ribose have been of particular interest.

[0003] Such 2' modifications have been shown to enhance the biophysical and pharmacological properties of the modified nucleic acids while maintaining key physicochemical principles of nucleic acid function, such as helical structure and base pair specificity, facilitating their widespread incorporation into nucleic acid therapeutics. Among these, 2'-fluoro (2'F), 2'-O-methyl (2'OMe), 2'-O-(2-methoxyethyl) (MOE), and 2',4'-locked, bridged, or constrained (e.g., tricyclo) nucleic acids have been widely studied. 1 .

[0004] 2'OMe is a natural RNA modification found in both the cap and body of human rRNA, tRNA, small nuclear RNA (snRNA), and human mRNA, and is therefore inherently biocompatible and unlikely to elicit the innate immune system. Indeed, 2'OMe modifications of viral RNA appear to be utilized by some viruses as a self-signal that allows them to evade interferon-mediated antiviral responses.

[0005] 2'OMe and related MOE modifications (Figures 1a, 4a) exhibit a variety of favorable physicochemical, pharmacological and immunological properties, and their clinical utility has been validated in recently approved nucleic acid medicines such as the silencing RNA (siRNA) drugs Patisiran and Givosiran (2'OMe) and the antisense oligonucleotide (ASO) drugs Nusinersen (Spinraza), Inotersen (Tegsedi) and Volanesorsen (Waylivra) (all MOEs). 2 Furthermore, 2'OMe-RNA modifications at purine bases have been found to be beneficial in Pegaptanib (Macugen), an FDA-approved aptamer drug for the treatment of age-related macular degeneration.

[0006] However, 2'OMe- and MOE-modified oligonucleotides are currently synthesized primarily by solid-phase phosphoramidite-based chemical synthesis, which is limited to short oligomers and a relatively small number of unique sequences, hindering their evolution. Therefore, applicable sequences of 2'OMe- and MOE-modified oligonucleotides to be screened for the desired therapeutic effect must be semi-rationally designed. Although this approach seems reasonable for ASO therapeutics designed to bind to regulatory sequences of messenger RNA, it impedes the novel discovery and development of aptamer and nucleic acid enzyme therapeutics in these important chemistries, slowing the development of nucleic acid nanotechnology objects and devices for both biotechnological and medical applications.

[0007] This allows the identification of mutant T7 RNA polymerase 3、4、5、6 or a mutant of the Stoffel fragment of Taq DNA polymerase 7 This has spurred the development of a variety of engineered polymerases as synthetic and reverse transcription tools, including those described in the literature, and has led to the discovery of partially and fully substituted 2'OMe-RNA aptamers. 6、8More recently, a mutant of KOD DNA polymerase has been shown to inhibit Mn 2+ reported that 1 kb 2'OMe-RNA fragments could be synthesized in the presence of ions, enabling the evolution of mixed LNA / 2'OMe-RNA aptamers against thrombin. 9 .

[0008] Despite these advances, enzymatic synthesis of bulkier MOE-RNAs has not been reported. Moreover, due to the outstanding importance and potential of 2'OMe-RNAs, tools for more efficient synthesis of longer or more complex 2'OMe-RNAs remain desirable. Summary of the Invention

[0009] (Summary of the invention) In an embodiment of the invention, there is provided a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, said nucleic acid polymerase comprising an amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO: 1, said amino acid sequence being mutated at T541 and / or K592 relative to the amino acid sequence of SEQ ID NO: 1. The amino acid sequence may be mutated at E664 relative to the amino acid sequence of SEQ ID NO: 1.

[0010] The amino acid sequence may comprise i) T541 mutation and K592 mutation, ii) T541 mutation and E664 mutation, or iii) T541 mutation, K592 mutation, and E664 mutation.T541 mutation may be T541G, T541S, T541A, T541C, T541D, T541P, or T541N.In certain embodiments, T541 mutation is T541G.K592 mutation may be K592G, K592A, K592C, K592M, K592S, K592D, K592P, K592N, K592T, K592E, K592V, K592Q, K592H, K592I, or K592L. In certain embodiments, the K592 mutation is K592A or K592G. The E664 mutation can be E664K or E664R.

[0011] In certain embodiments, the amino acid sequence comprises the mutations T541G and K592A.

[0012] The amino acid sequence may include one or more, or all of the following mutations relative to SEQ ID NO: 1: V93Q, D141A, E143A, and A485L. The amino acid sequence may include one or more, or all of the following mutations relative to SEQ ID NO: 1: Y409, I521, and F545. The amino acid sequence may include one or more, or all of the following mutations relative to SEQ ID NO: 1: Y409G, I521L or I521H, and F545L.

[0013] The amino acid sequence may include a D614 mutation relative to SEQ ID NO: 1. The D614 mutation may be D614N.

[0014] The amino acid sequence may have at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO: 1. The amino acid sequence may have at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, where residues 93, 141, 143, 409, 485, 521, 541, 545, 592, and 664 are unchanged. The amino acid sequence may have at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, where residues 93, 141, 143, 409, 485, 521, 541, 545, 592, 614, and 664 are unchanged.

[0015] The amino acid sequence may comprise SEQ ID NO:7 or SEQ ID NO:8.

[0016] In another aspect of the invention, there is provided a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, the nucleic acid polymerase comprising an amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence being mutated at E664R relative to the amino acid sequence of SEQ ID NO: 1. The nucleic acid polymerase can comprise any feature, sequence, mutation, characteristic, or pattern of mutations as disclosed herein for nucleic acid polymerases.

[0017] The nucleic acid polymerases disclosed herein can comprise an amino acid sequence that includes one or more, or any combination, of the following mutations relative to SEQ ID NO:1: D540, D542, K591, K593, Y663, and Q665.

[0018] In another aspect of the invention, there is provided a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, comprising an amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at one, all, or any combination of positions D540, D542, K591, K593, Y663, and / or Q665 relative to SEQ ID NO:1.

[0019] In some embodiments, the mutation at D540 is D540A, D540G, D540S, or D540C. In particular, the mutation can be D540A. In some embodiments, the mutation at D542 is D542A, D542G, D542S, or D542C. In some embodiments, the mutation at K591 is K591G, K591A, K591C, K591M, K591S, K591D, K591P, K591N, K591T, K591E, K591V, K591Q, K591H, K591I, or K591L. In some embodiments, the mutation in K593 is K593G, K593A, K593C, K593M, K593S, K593D, K593P, K593N, K593T, K593E, K593V, K593Q, K593H, K593I, or K593L. In some embodiments, the E663 mutation can be E663K, E663R, or E663H. In some embodiments, the E665 mutation can be E665K, E665R, or E665H.

[0020] The nucleic acid polymerases disclosed herein are capable of producing a non-DNA nucleotide polymer from a nucleic acid template, where the non-DNA nucleotide polymer comprises 2'-O-methyl-RNA and (2'OMe-RNA) nucleotides and / or 2'-O-(2-methoxyethyl)-RNA (MOE-RNA) nucleotides.

[0021] The nucleic acid polymerases disclosed herein may have an amino acid sequence derived from the wild-type sequence of a polB family nucleic acid polymerase. The nucleic acid polymerases disclosed herein may have an amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:9.

[0022] In another aspect of the invention, a method of making a non-DNA nucleotide polymer is provided, comprising contacting a nucleic acid template with a nucleic acid polymerase of any one of the preceding claims under conditions conducive to polymerization. In some embodiments, 2'OMe-RNA and / or MOE-RNA nucleotides are provided during polymerization and the resulting non-DNA nucleotide polymer comprises said nucleotides.

[0023] In another aspect of the present invention, there is provided a use of any of the nucleic acid polymerases disclosed herein for the production of a non-DNA nucleotide polymer. In some embodiments, the non-DNA nucleotide polymer comprises 2'OMe-RNA nucleosides and / or MOE-RNA nucleosides.

[0024] In another aspect of the invention, a nucleic acid encoding any of the polymerases disclosed herein is provided.

[0025] In another aspect of the invention, a host cell is provided that contains any of the polymerases disclosed herein or any nucleic acid encoding a polymerase disclosed herein. [Brief description of the drawings]

[0026] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1. Two-residue steric gate. a) Chemical structure of 2'-O-methyl (2'OMe)-RNA. The 2'-methoxy substituent is highlighted in cyan. b) Sequence alignment of wild-type and engineered polymerase Tgo with the key mutations TGK (blue), TGLLK (green) and 2M (red). The sequences shown in panel b) are SEQ ID NOs: 10, 11, 12, and 13. c) Space-filling model of the ternary structure of KOD DNA polymerase (PDB ID 5OMF) with the mutations TGK (blue), TGLLK (green) and 2M (red). d) Structural model of the active site of KOD DNA polymerase (PDB ID 5OMF) with DNA template strand (orange), active site 2'OMe-ATP, and 2'OMe-RNA nascent strand (cyan), with the 3'-terminal 2'-methoxy group and +1 nucleotide shown as space-filling envelopes, the key steric gate mutations (T541G, K592A) in pink (sticks), and wild-type side chain residues shown as space-filling envelopes to highlight reduced steric volume. e) Denaturing PAGE of 2'OMe-RNA synthesis (DNA primer FD, template TempNpure, total length +72nt) with single and double steric gate mutations. Note the synergistic effect of the T541G and K592A double mutations. f-h) Denaturing PAGE of DNA (H), RNA (OH) and 2'OMe-RNA (OMe) synthesis by TGK, TGLLK or 2M on f) defined sequence template (DNA / 2'OMe-RNA primer FD, template TempNpure, total length +72nt), g) random N40 template (RNA / 2'OMe-RNA primer A-Test2, template Tag3.3-N40-Test2, total length +79nt), densitometry of N40 synthesis yields: TGLLK 2'OMe-RNA 0%, 2M 2'OMe-RNA 90% (SI Figure 17), and h) long-range synthesis of GFP transcript (2'OMe-RNA primer Synth-out1mm, template sfGFP, total length +752nt). [Diagram 2]Figure 2. Site-specific RNA endonuclease catalysts composed of 2'OMe-RNA. Sequences and predicted secondary structures of a) 2'OMezyme R15 / 5-K selected to target RNA "Sub_KRas_12" [G12D] (residues 213-242 of human KRAS mRNA harboring the c.35G>A (G12D) mutation (SEQ ID NOs: 14 and 15)) and b) mutant 2'OMezyme R15 / 5-C retargeted to alternative RNA "Sub_CTNNB1_33" (residues 85-111 of human CTNNB1 mRNA harboring the c.98C>A (S33Y) mutation). 2'OMe-RNA nucleotides are shown in cyan or blue (residues changed from R15 / 5-K to R15 / 5-C) and RNA substrates are shown in orange (KRAS) or red (CTNNB1). Black arrows indicate the RNA cleavage site. Circled residues indicate bases in the "R15_1" parent 2'O Mezyme that were changed during reselection (bottom) (SEQ ID NOs: 16 and 17). c, d) (Left panel) Urea PAGE gel shows 2'O Mezyme (5 μM) performing allele-specific cleavage of substrate RNA (1 μM) Sub_KRas_12 and Sub_CTNNB1_33 in a bimolecular reaction in trans under quasi-physiological conditions (37 °C, pH 7.4, 1 mM Mg2+, 17.5 h). Lane 1 shows partially hydrolyzed RNA substrate. (Right panel) Graph shows pre-steady-state single turnover reaction with substrate RNA (1 μM), 2'O Mezyme (5 μM) and the indicated reaction conditions at 37 °C. Error bars indicate standard deviation (sem) of the mean of three independent replicates. e&f) Reaction of (5 μM) 2'O Mezyme and (0.5 μM) synthetic RNA transcripts of e) KRAS ("Sub_KRas_ORF") and f) CTNNB1 ("Sub_CTNNB1_ORF") carrying the indicated mutations under sub-physiological conditions (37°C, pH 7.4, 1 mM Mg2+, 65 h). [Diagram 3]Figure 3. MOE-RNA synthesis. a) Chemical structure of 2'-O-(2-methoxyethyl)-RNA (MOE-RNA) with the 2'-O-(2-methoxyethyl) group highlighted. b) Ribose sugar puckering equilibrium. The 2'-O-MOE modification shifts the equilibrium to a C3'-endo (N-form) conformation comparable to RNA. c) Side-on (left) and top-on (right) space-filling representations of the X-ray structure of the MOE-RNA duplex (PDB ID 468D) with the 2'-O-(2-methoxyethyl) group (highlighted), as well as a superposition of the observed 2'-O-(2-methoxyethyl) conformation next to a Newman projection of ethylene glycol monomethyl ether, which preferentially adopts a gauche conformation at each of the two oxygen atoms (stick diagram, center). d-f) Denaturing PAGE of 2'OMe-RNA (OMe) and MOE-RNA (MOE) synthesis by TGLLK or 2M on d) a defined sequence template (2'OMe-RNA primer FD, template TempNpure, total length +72nt), e) a random N40 template (2'OMe-RNA primer A-Test2, template Tag3.3-N40-Test2, total length +79nt), densitometry of N40 synthesis yields: TGLLK 2'OMe-RNA 1%, TGLLK MOE-RNA 0%, 2M 2'OMe-RNA 84%, 2M MOE-RNA 65% (SI Figure 17), and f) long-range synthesis of GFP transcript (2'OMe-RNA primer Synth-out1mm, template sfGFP, total length +752nt). [Figure 4] Figure 4. 2'OMe / MOE-RNA aptamers and binding kinetics. a-c) Representation of the sequence and secondary structure of the anti-VEGF aptamer ARC2246 (upper panel), their respective SPR sensorgrams and average KD (middle), and residues of fitted curves (bottom) for a) ARC224 2'OMe-GACU, b) ARC224 2'OMe-GU MOE-AC (MOE substitution, green) and c) ARC224 2'OMe-U MOE-ACG (SPR binding kinetics: Supplementary Table 3). Sequences are SEQ ID NOs: 18-20. [Diagram 5]Figure 5. Nascent strand steric gate and polymerase motifs. a) Conserved sequence motifs in the polB polymerase family showing the sequence context and conservation of the nascent strand steric gate of motif C (T541) and motif KxY (K592). b) Structural context of the steric gate with D540 and the active site 2'OMe-ATP (KOD DNA polymerase (PDB ID 5OMF)) showing direct contact to the +1 minor groove and indirect contact (via HO) to the 3'-terminal nucleotide. c) Structural conservation of the nascent strand steric gate across the polB phylogeny from archaeal polB polymerases (left), to bacterial polB polymerases (middle) to eukaryotic polB polymerases (right). [Figure 6] Figure 6 (Supplementary Figure 1) Polymerase screening. a) Sequence alignment showing the engineered polymerases and the respective key mutations in TGLLK (blue and green), TGHLK (orange) and 2M (red). Sequences are SEQ ID NOs: 12, 21, and 13. b) Representation of the relative positions of the screened residues (D540, T541, K592, D614, E664) in the polymerase structure (KOD DNA polymerase (PDB ID 5OMF)) using the polymerase activity assay (PAA) described in Materials & Methods. c) Denaturing PAGE of 2'OMe-RNA synthesis by different TGLLK (I521L) single mutants identified in the screen on a defined sequence template (DNA primer FD, template TempNpure, total length +72nt). Note the positive effect of T541G and K592A and E664R mutations. In this context, we also investigated the mutation to L521H in the TGLLK context, which enhances 2'OMe-RNA synthesis but ultimately favors the I521L mutant. d) Denaturing PAGE of 2'OMe-RNA synthesis by different TGLLK and TGHLK mutants on a defined sequence template (DNA primer FD, template TempNpure, total length +72nt). Note the synergistic effect of the T541G and K592A double mutations. e) Denaturing PAGE of DNA / 2'OMe-RNA synthesis by 2M on a random N40 template (DNA / 2'OMe-RNA primer A-Test2, template Tag3.3-N40-Test2, total length +79nt). [Figure 7]Figure 7 (Supplementary Figure 2) pH and magnesium dependence of 2'Omezyme R15 / 5-K. Normalized activity of 2'Omezyme R15 / 5-K (5 μM) or the analogous DNAzyme "1023_KrasC" (5 μM) on Sub_Kras_12[G12D] RNA (1 μM) at varying pH in the buffer systems as indicated (1 mM Mg2+, 37 °C, 16.5 h) or (b) varying concentrations of MgCl2 (pH 7.4, 37 °C, 16.5 h). (c) Pre-steady-state single-turnover reaction with substrate RNA Sub_Kras_12[G12D] RNA (1 μM) and 2'Omezyme R15 / 5-Kras (5 μM) in the absence of Mg2+ (pH 7.4, 37 °C, 5 mM EDTA). Error bars indicate standard error of the mean (sem) of three independent replicates. (d & e) Urea PAGE gel showing (10 nM) 2'Omezymes (d) R15 / 5-K or (e) R15 / 5-C subjected to multi-turnover catalysis with (d) Sub_KRas_12[G12D] or (e) Sub_CTNNB1_33[S33Y] of (1 μM) RNA substrate under quasi-physiological conditions (37 °C, pH 7.4, 1 mM Mg2+). [Figure 8] Figure 8 (Supplementary Figure 3). Characterization of 2'O Mezyme R15 / 5-K-catalyzed RNA cleavage products. (a) MALDI-ToF spectrum of the 5' RNA product of R15 / 5-K-catalyzed cleavage of RNA Sub_KRas_12[G12D]. The expected mass of the product is indicated with the 3' monophosphate (p) or cyclic phosphate (>p) (as shown in the schematic) (SEQ ID NO: 22). (b) Phosphatase assay of the 5' product of R15 / 5-K-catalyzed cleavage of RNA Sub_KRas_12[G12D]. Urea PAGE gel showing PAGE-purified 5' product RNA treated with or without prior acid hydrolysis with calf intestinal phosphatase (CIP; removes 2' or 3' terminal monophosphate but not 2',3'-cyclic phosphate) or T4 polynucleotide kinase (T4 PNK; removes both monophosphate and 2',3'-cyclic phosphate). Lane 1 shows the partially hydrolyzed RNA substrate as a marker. [Figure 9]Figure 9 (Supplementary Figure 4). Serum nuclease resistance of 2'OMezyme R15 / 5-K. (a) Urea-PAGE gel and graph showing the stability of 2'OMezyme R15 / 5-K and the similar DNAzyme "1023_KRasC" in 90% human serum at 37 °C. (b) Urea-PAGE gel showing the activity of (5 μm) 2'OMezyme R15 / 5-K in reaction with the RNA substrate Sub_KRas_12[G12D] (1 μM) under quasi-physiological conditions (pH 7.4, 1 mM Mg2+, 37 °C, 18 h) before (lane 3) or after (lane 4) incubation in 90% human serum for 120 h at 37 °C. Lane 1 shows the partially hydrolyzed RNA substrate as a marker. [Figure 10] Figure 10 (Supplementary Figure 5). Mutational screening of putative unpaired substrate-proximal nucleobases in retargeting 2'OMezyme R15 / 5-CTNNB1. (a) Sequence and predicted secondary structure of retargeting 2'OMezyme "R15 / 5-CTNNB1" bound to RNA substrate "Sub_CTNNB1_33" (residues 85-111 of human CTNNB1 mRNA harboring the c.98C>A (S33Y) mutation). 2'OMe-RNA nucleotides are shown in cyan or blue (indicating sequence changes from R15 / 5-K) or orange (indicating changes from the parent R15 / 5_1 2'OMezyme), and the RNA is shown in orange. Black arrows indicate the RNA cleavage site. Mutants of 2'OMezyme were prepared with all possible single mutations (or one double mutation, A39G + U45A) of the putative unpaired positions adjacent to the substrate binding arm, as indicated by circles. Sequences shown are SEQ ID NOs: 16 and 23. (b) Urea PAGE gel showing the activity of mutants of R15 / 5-CTNNB1 (2.5 μM) against the RNA substrate Sub_CTNNB1_33[S33Y] (1 μM) under sub-physiological conditions (pH 7.4, 1 mM Mg2+, 37° C., 24 h). The R15 / 5-CTNNB1: A39G, U45A mutant (referred to as *R15 / 5-C) was used for all other experiments. [Figure 11]Figure 11 (Supplementary Figure 6): General synthetic pathway for the triphosphorylation of 2'-O-(2-methoxyethyl)ribonucleosides. Base = adenine (A, compound a), 5-methyluracil (mU, compound b), guanine (G, compound c), or cytosine (C, compound d). i) POCl3, proton sponge, (MeO)3PO, -15 °C; ii) (Bu4N)3HP2O7, Bu3N, DMF, RT, 30 min; iii) TEAB buffer, RT, 13–28% over three steps (one-pot). [Figure 12] Figure 12 (Supplementary Figure 7) Time course of 2'OMe-RNA and MOE-RNA synthesis. a) Denaturing PAGE of the time course of 2'OMe-RNA and MOE-RNA synthesis by TGLLK and 2M on a defined sequence template (2'OMe-RNA primer FD, template TempNpure, full length +72nt). 2M reaches full length synthesis (+72nt) in less than 5 minutes (2'OMe-RNA) and 20 minutes (MOE-RNA), respectively. b) Denaturing PAGE of the time course of DNA, 2'OMe-RNA, and MOE-RNA synthesis by 2M on a random N40 sequence template (2'OMe-RNA primer FD-Test2, template Tag3.3-N40-Test2, full length +79nt). 2M achieves full-length synthesis (+79 nt) in less than 1 min (DNA), 10 min (2'OMe-RNA), and 30 min (MOE-RNA, densitometry measurements in SI Fig. 17 ), respectively. [Figure 13] Figure 13 (Supplementary Figure 8) Synthesis of 2'OMe-RNA, mixed 2'OMe / MOE-RNA, and total MOE-RNA. (From left to right) Denaturing PAGE of 2'OMe-RNA synthesis by TGLLK and 2M on a defined sequence template (2'OMe-RNA primer FD, template TempN, full length +57nt), mixed 2'OMe / MOE-RNA synthesis (2'OMe-U / G / C MOE-A, 2'OMe-G / C MOE-A / m5U, 2'OMe-C MOE-A / m5U / G), and total MOE-RNA synthesis. Note that the gel shift (retardation) increases with increasing MOE content, indicating an increased hydrodynamic envelope of the 2'-O-(2-methoxyethyl) groups protruding from the helix. [Figure 14]Figure 14 (Supplementary Figure 9) 2'OMe / MOE-RNA aptamers. a), b) Sequence and secondary structure representation of anti-VEGF aptamer ARC22413 for a) ARC224 2'OMe- and ARC224 2'OMe m5U and b) ARC224 MOE (top panel), respective SPR sensorgrams and average KD (middle), residues of fitted curve (bottom). Note the reduced affinity of ARC224 2'OMe-m5U compared to ARC224 (2'OMe-U). Sequences are SEQ ID NOs: 24 and 25. [Figure 15] Figure 15. (Supplementary Figure 10) Polymerase phylogeny and motif conservation. a) Phylogenetic tree of polB family polymerases including archaeal (Pyrococcales / Thermococcales), bacterial (E. coli, RB69 bacteriophage), eukaryotic (Saccharomyces), mammalian (human), and viral (Vaccinia) polymerases. b) Sequence alignment between different polB polymerases and conservation of motifs C (left) and KxY (right). Sequences are SEQ ID NOs: 26-40. [Figure 16] Figure 16 (Supplementary Figure 11) Fidelity of MOE-RNA synthesis by 2M. Dropout assay of MOE-RNA fidelity showing templated synthesis of the first four bases on the TempNpure template (3'-CTAG-5' after the priming site) omitting one MOE-NTP (from left to right: MOE-CTP, MOE-GTP, MOE-m5UTP, MOE-ATP) showing the expected termination pattern for correct incorporation, except for MOE-GTP showing some misincorporation on the opposite template C. Full-length synthesis (+72nt) by all MOE-NTPs is also shown. [Figure 17]Figure 17 (Supplementary Figure 12) Steady-state kinetics for 2'OMe-RNA primer extension with ATP, 2'OMe-ATP and MOE-ATP by 2M. a) Steady-state kinetic parameters V0 (μmoles / min) plotted against nucleotide triphosphate concentration [NTP] for extension of 2'OMe-RNA primer FAM-FD on template BFL770 (Supplementary Table 1) by 2M (n=3) for ATP (black circles), 2'OMe-ATP (red squares) and MOE-ATP (cyan triangles). b) Table of steady-state kinetic parameters for single base incorporation by 2M. [Figure 18] Figure 18 (Supplementary Figure 13) Evaluation of 2M against other polymerases. a) Denaturing PAGE of RNA, 2'F-DNA, and 2'OMe-RNA synthesis by 2M and engineered Taq Stoffel fragment mutant SFM4-6 on a defined sequence template (DNA or 2'OMe-RNA primer FD, template TempNpure, full length +72nt) under optimal conditions for each polymerase. b) Denaturing PAGE of RNA and 2'OMe-RNA transcription by T7 RNA polymerase (WT) and engineered T7 RNAP mutant RGVG-M6 on a long defined sequence template (made as described in Materials & Methods, 901 bp) under optimal conditions for RGVG-M6. c) Denaturing PAGE of 2'OMe-RNA primer extension synthesis and transcription by 2M and engineered T7 RNAP mutant RGVG-M6 with a long defined sequence template (for transcription reaction: template prepared as described in Materials & Methods, 901 bp; for primer extension reaction: 2'OMe-RNA primer Synthout1mm, template sfGFP, full length +752 nt) under equimolar nucleic acid input (50 nM (0.5 pmol) input of primer and dsDNA template) in the presence and absence of 1.5 mM Mn2+. [Figure 19]Figure 19 (Supplementary Figure 14) Comparison of polymerases. Denaturing PAGE of 2'OMe- and MOE-RNA synthesis by 2M, engineered KOD mutant DGLNK14, and 2M carrying DGLNK mutation D614N (2M D614N) on a defined sequence template (2'OMe-RNA primer FD, template TempNpure, full length +72nt) both in the presence and absence of Mn2+ ions under optimal conditions for each polymerase. As described14, KOD DGLNK performs best in 2'OMe-RNA synthesis in the presence of Mn2+, but is unable to synthesize MOE-RNA efficiently. Interestingly, the D614N mutation slightly increases activity for 2M in the context of 2'OMe-RNA synthesis. [Figure 20] Figure 20 (Supplementary Figure 15) Polymerase comparison of 2M vs. 3M. a) Sequence alignment showing the key mutations in polymerase Tgo wild type and engineered polymerases and TGK (blue), TGLLK (green) and 2M (red) and 3M (brown), respectively. SEQ ID NOs: 10, 11, 12, 13, 41. b) Denaturing PAGE of DNA (H), RNA (OH), 2'F-RNA (F) and 2'OMe-RNA (OMe) synthesis by TGK, TGLLK, 2M and 3M on a defined sequence template (DNA / 2'OMe-RNA primer FD, template TempNpure, total length +72nt). c) Denaturing PAGE of 2'OMe-RNA (OMe) and MOE-RNA (MOE) synthesis by TGLLK, 2M and 3M on a defined sequence template (2'OMe-RNA primer FD, template TempNpure, total length +72nt). [Figure 21]Figure 21 (Supplementary Figure 16) 2'OMezyme R15 / 5-C as an analogue of a hairpin ribozyme. a) Sequence and predicted secondary structure of 2'OMezyme R15 / 5-C engineered to target human CTNNB1 mRNA RNA (top) and hairpin ribozyme (Hpz) (bottom). 2'OMe-RNA nucleotides (R15 / 5-C) are shown in orange or cyan (mutated or identical to the Hpz consensus). RNA nucleotides are shown in red or cyan (if equivalent to R15 / 5-C). RNA substrate is shown in grey. Black arrows indicate the RNA cleavage site. Sequences are SEQ ID NOs: 42, 43, 44. b) Urea PAGE gel showing cleavage of Sub_CTNNB1_33 substrate RNA (1 μM) by mutants of R15 / 5-C with mutations relative to the Hpz consensus. (c) Urea PAGE gel showing the RNA ligation activity of 2'O Mezyme R15 / 5_1. PAGE purified 5' (FITC-labeled) and 3' (unlabeled) RNA cleavage products of R15 / 5-K-catalyzed cleavage of Sub_KRas_12[G12D] (1 μM each) re-incubated with R15 / 5-K (5 μM) at -7 °C on ice (lanes 2-5) or supercooled (lanes 7-10) for 20 h in sub-physiological buffer (pH 7.4, 1 mM Mg2+) (lanes 4, 5, 9, 10, 12 and 13) or magnesium-free buffer (pH 7.4, 5 mM EDTA) (lanes 2, 3, 7, 8) or at 37 °C. Lane 1 shows partially hydrolyzed RNA Sub_KRas_12[G12D] substrate as a marker. [Figure 22] Figure 22 (Supplementary Figure 17) a) Densitometry measurements of DNA, 2'OMe-RNA, and MOE-RNA synthesis over time by 2M in the N40 library (SI Figure 7b), and b) synthesis yields of 2'OMe-RNA and MOE-RNA by TGLLK and 2M in the N40 library (Figures 1g and 3e). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description of the Invention Provided herein are polymerases that may contain mutations in a two-residue stereocontrol "gate". The polymerases provided herein have been engineered to reduce the steric bulk of this gate, resulting in an increased ability of the polymerase to synthesize xenonucleic acid (XNA) polymers. In particular, the polymerases may be capable of incorporating 2'-O-methyl-RNA and (2'OMe-RNA) nucleotides and / or 2'-O-(2-methoxyethyl)-RNA (MOE-RNA) nucleotides into the polymer.

[0028] Thus, in one embodiment, there is provided a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, the nucleic acid polymerase comprising an amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence is mutated at T541 and / or K592 relative to the amino acid sequence of SEQ ID NO: 1. In other words, the polymerase may be mutated at i) T541, ii) K592, or iii) T541 and K592 relative to the amino acid sequence of SEQ ID NO: 1.

[0029] The polymerase may comprise an E664 mutation relative to SEQ ID NO:1.

[0030] In some embodiments, the nucleic acid polymerase comprises mutations at T541 and K592. In some embodiments, the nucleic acid polymerase comprises mutations at T541 and E664. In some embodiments, the nucleic acid polymerase comprises mutations at T541, K592, and E664.

[0031] The mutations at T541 and / or K592 may be to less bulky residues. Thus, the mutations may be to any residue that is less sterically blocking than threonine at position 541 or lysine at position 592. The T541 mutation may be selected from the group T541G, T541S, T541A, T541C, T541D, T541P, or T541N. In particular, the T541 mutation may be T541G or T541S. The K592 mutation may be K592G, K592A, K592C, K592M, K592S, K592D, K592P, K592N, K592T, K592E, K592V, K592Q, K592H, K592I, or K592L. In particular, the K592 mutation may be K592G, K592A, K592C, or K592M.

[0032] The mutation at E664 may be a mutation to any positively charged residue. The E664 mutation may be E664K, E664R, or E664H. In particular, the E644 mutation may be E664K or E664R.

[0033] In an embodiment, the mutation in T541 is T541G. In an embodiment, the mutation in K592 is K592A or K592G. In an embodiment, the mutation in E644 is E664K or E664R. The polymerase may comprise mutations T541G and K592A. The polymerase may comprise mutations T541G and E664K. The polymerase may comprise mutations T541G and E664R. The polymerase may comprise mutations T541G, K592A, and E664K. The polymerase may comprise mutations T541G, K592A, and E664R.

[0034] The polymerase may comprise the mutation T541G and a mutation at position K592. The mutation at position K592 may be any disclosed herein, such as A or G. The polymerase may comprise the mutation T541G, a mutation at position K592, and a mutation at position E664.

[0035] The polymerase may comprise a mutation at any one, all, or any combination of positions D540, D542, K591, K593, Y663, and / or Q665 relative to SEQ ID NO: 1. Optionally, the mutation at positions D540, D542, K591, and / or K593 is to any less bulky residue, i.e., any residue that is less sterically blocking than the wild-type residue. Optionally, the mutation at positions Y663 and / or Q665 is to any positively charged residue.

[0036] In some embodiments, the mutation at D540 is D540A, D540G, D540S, or D540C. In particular, the mutation may be D540A.

[0037] In some embodiments, the mutation at D542 is D542A, D542G, D542S, or D542C.

[0038] In some embodiments, the mutation at K591 is K591G, K591A, K591C, K591M, K591S, K591D, K591P, K591N, K591T, K591E, K591V, K591Q, K591H, K591I, or K591L.

[0039] In some embodiments, the mutation at K593 is K593G, K593A, K593C, K593M, K593S, K593D, K593P, K593N, K593T, K593E, K593V, K593Q, K593H, K593I, or K593L.

[0040] In some embodiments, the E663 mutation can be E663K, E663R, or E663H.

[0041] In some embodiments, the E665 mutation can be E665K, E665R, or E665H.

[0042] In certain embodiments, a nucleic acid polymerase capable of generating a non-DNA nucleotide polymer from a nucleic acid template is provided, the nucleic acid polymerase comprising an amino acid sequence having at least 36% identity with the amino acid sequence of SEQ ID NO: 1, the amino acid sequence being mutated at T541 and K592 relative to the amino acid sequence of SEQ ID NO: 1. In an embodiment, the nucleic acid polymerase comprises mutations T541G and K592A / K592G. In an embodiment, the nucleic acid polymerase comprises mutations T541G and K592A.

[0043] In another embodiment, there is provided a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, comprising an amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence is mutated at T541 and K592 relative to the amino acid sequence of SEQ ID NO:1, e.g., T541G and K592A / K592G, and wherein the amino acid sequence is mutated at one or more of positions D540, D542, K591, K593, Y663, and / or Q665 relative to SEQ ID NO:1, or any combination, relative to the amino acid sequence of SEQ ID NO:1.

[0044] In another embodiment, a nucleic acid polymerase capable of generating a non-DNA nucleotide polymer from a nucleic acid template is provided, the nucleic acid polymerase comprising an amino acid sequence having at least 36% identity with the amino acid sequence of SEQ ID NO: 1, the amino acid sequence being mutated at T541, K592, and E644 relative to the amino acid sequence of SEQ ID NO: 1. In an embodiment, the nucleic acid polymerase comprises mutations T541G, K592A / K592G, and E664K / E664R. In an embodiment, the nucleic acid polymerase comprises mutations T541G, K592A, and E664K. In another embodiment, the nucleic acid polymerase comprises mutations T541G, K592A, and E664R.

[0045] In another embodiment, there is provided a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, comprising an amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence is mutated at T541, K592, and E644 relative to the amino acid sequence of SEQ ID NO:1, e.g., T541G, K592A / K592G, and E664K / E664R, and wherein the amino acid sequence is mutated at one or more of positions D540, D542, K591, K593, Y663, and / or Q665 relative to SEQ ID NO:1, or any combination, relative to the amino acid sequence of SEQ ID NO:1.

[0046] Both T541 and K592 are part of motifs (motifs C and KxY, respectively) that are very highly conserved at both the sequence and structural levels (Figure 5, SI Figure 10) in polB polymerases of archaeal, eukaryotic and even viral origin (Kazlauskas et al., Diversity and evolution of B-family DNA polymerases, Nucleic Acids Res 2020, 48(18): 620 10142-10156). Therefore, the mutations of the present disclosure can be applied to the polymerase sequence of any polymerase from the polB family or can be derived from any polymerase from the polB family. In certain embodiments, the backbone is any polB polymerase. In other embodiments, the backbone is any polB polymerase except viral polymerases. The backbone can be from the archaeal Thermococcus and / or Pyrococcus polymerases.

[0047] The polymerase can be a mutant of the polymerase from T. goronarius (Tgo). The sequence of wild-type Tgo is shown below: [ka] (Sequence number 1).

[0048] Any of the nucleic acid polymerases disclosed herein may comprise an amino acid sequence having at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO: 1. The amino acid sequence may have at least 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO: 1. The amino acid sequence may be mutated at T541 and / or K592, and optionally E664, relative to the amino acid sequence of SEQ ID NO: 1. The polymerase may comprise any specific mutation or pattern of mutations as disclosed herein.

[0049] The polymerases disclosed herein can include a V93 mutation relative to SEQ ID NO: 1. The mutation can be V93Q.

[0050] The polymerases disclosed herein can include a D141 mutation and / or an E143 mutation relative to SEQ ID NO: 1. The mutation can be D141A and / or E143A.

[0051] The polymerases disclosed herein can include an A485 mutation relative to SEQ ID NO: 1. The mutation can be A485L.

[0052] The amino acid sequence of the nucleic acid polymerase may further comprise one or more, or all of the following mutations: V93Q, D141A, E143A, and A485L.

[0053] V93Q is a mutation known to abolish uracil termination, D141A and E143A reduce 3'-5' exonuclease function, and the "therminator" mutation (A485L) is known to enhance incorporation of unnatural substrates. The sequence of Tgo polymerase (hereafter referred to as TgoT) containing these mutations is shown below: [ka] (Sequence number 2).

[0054] The mutations of any of the embodiments disclosed herein, in which the mutations are applied to a backbone comprising SEQ ID NO:1, may be applied to a backbone comprising SEQ ID NO:2, in which residues 93, 141, 143, and 485 are invariant. For example, in some embodiments, a nucleic acid polymerase capable of generating a non-DNA nucleotide polymer from a nucleic acid template is provided, the nucleic acid polymerase comprising an amino acid sequence having at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO:2, the amino acid sequence being mutated at T541 and / or K592, and optionally E664, relative to the amino acid sequence of SEQ ID NO:1, in which residues 93, 141, 143, and 485 are invariant. The amino acid sequence may also comprise a mutation at any one or any combination of positions D540, D542, K591, K593, Y663, and / or Q665.

[0055] The polymerases disclosed herein can include a Y409 mutation relative to SEQ ID NO: 1. In some cases, the Y409 mutation can be Y409N or Y409G.

[0056] The polymerases disclosed herein can include an I521 mutation relative to SEQ ID NO: 1. In some cases, the I521 mutation can be I521L or I521H (see Figure 6 (Supplementary Figure 1)).

[0057] The polymerases disclosed herein can include an F545 mutation relative to SEQ ID NO: 1. In some cases, the F545 mutation can be F545L.

[0058] The polymerases disclosed herein can include a D614 mutation relative to SEQ ID NO: 1. In some cases, the D614 mutation can be D614N (see Figure 19 (Supplementary Figure 14)).

[0059] The polymerase may comprise mutations Y409, I521, T541G, F545, K592A / K592G, and E664 relative to SEQ ID NO: 1 or SEQ ID NO: 2. The polymerase may comprise mutations Y409G / Y409N, I521L / I521H, T541, F545L, K592, and E664K / E664R relative to SEQ ID NO: 1 or SEQ ID NO: 2. The polymerase may comprise mutations Y409G / Y409N, I521L / I521H, T541G, F545L, K592A / K592G, and E664K / E664R relative to SEQ ID NO: 1 or SEQ ID NO: 2. The polymerase may comprise the mutations Y409G, I521L, T541G, F545L, K592A, and E664K relative to SEQ ID NO: 1 or SEQ ID NO: 2. The polymerase may comprise the mutations V93Q, D141A, E143A, Y409G, A485L, I521L, T541G, F545L, K592A, and E664K relative to SEQ ID NO: 1. The polymerase may comprise the mutations Y409G, I521L, T541G, F545L, K592A, and E664R relative to SEQ ID NO: 1 or SEQ ID NO: 2. The polymerase can include the mutations V93Q, D141A, E143A, Y409G, A485L, I521L, T541G, F545L, K592A, and E664R relative to SEQ ID NO:1.

[0060] The polymerase may comprise the mutations Y409, I521, T541G, F545, K592A / K592G, D614N, and E664 relative to SEQ ID NO: 1 or SEQ ID NO: 2. The polymerase may comprise the mutations Y409G / Y409N, I521L / I521H, T541, F545L, K592, D614, and E664K / E664R relative to SEQ ID NO: 1 or SEQ ID NO: 2. The polymerase may comprise the mutations Y409G / Y409N, I521L / I521H, T541G, F545L, K592A / K592G, D614N, and E664K / E664R relative to SEQ ID NO: 1 or SEQ ID NO: 2. The polymerase may comprise the mutations Y409G, I521L, T541G, F545L, K592A, D614N, and E664K relative to SEQ ID NO: 1 or SEQ ID NO: 2. The polymerase may comprise the mutations V93Q, D141A, E143A, Y409G, A485L, I521L, T541G, F545L, K592A, D614N, and E664K relative to SEQ ID NO: 1. The polymerase may comprise the mutations Y409G, I521L, T541G, F545L, K592A, D614N, and E664R relative to SEQ ID NO: 1 or SEQ ID NO: 2. The polymerase can include the mutations V93Q, D141A, E143A, Y409G, A485L, I521L, T541G, F545L, K592A, D614N, and E664R relative to SEQ ID NO:1.

[0061] In certain embodiments, the nucleic acid polymerase comprises or can be of the following amino acid sequence: [ka] (SEQ ID NO:3; also known as 2M polymerase).

[0062] Thus, in embodiments there is provided a polymerase having an amino acid sequence having at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% similarity or identity to the amino acid sequence of SEQ ID NO:3, wherein residues 93, 141, 143, 409, 485, 521, 541, 545, 592 and 664 are unchanged (i.e. the mutations V93Q, D141A, E143A, Y409G, A485L, I521L, T541G, F545L, K592A and E664K are maintained).

[0063] In certain embodiments, the nucleic acid polymerase comprises or can be of the following amino acid sequence: [ka] (SEQ ID NO:4; also known as 3M polymerase).

[0064] Thus, in one aspect, there is provided a polymerase having an amino acid sequence having at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO:4, wherein residues 93, 141, 143, 409, 485, 521, 541, 545, 592, and 664 are unchanged (i.e., the mutations V93Q, D141A, E143A, Y409G, A485L, I521L, T541G, F545L, K592A, and E664R are maintained).

[0065] In certain embodiments, the nucleic acid polymerase comprises or can be of the following amino acid sequence: [ka] (SEQ ID NO:5; also known as 2M+D614N polymerase).

[0066] Thus, in embodiments there is provided a polymerase having an amino acid sequence having at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% similarity or identity to the amino acid sequence of SEQ ID NO:5, wherein residues 93, 141, 143, 409, 485, 521, 541, 545, 592, 614 and 664 are unchanged (i.e. the mutations V93Q, D141A, E143A, Y409G, A485L, I521L, T541G, F545L, K592A, D614N and E664K are maintained).

[0067] In certain embodiments, the nucleic acid polymerase comprises or can be of the following amino acid sequence: [ka] (SEQ ID NO:6; also known as 3M+D614N polymerase).

[0068] Thus, in embodiments there is provided a polymerase having an amino acid sequence having at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% similarity or identity to the amino acid sequence of SEQ ID NO:6, wherein residues 93, 141, 143, 409, 485, 521, 541, 545, 592, 614 and 664 are unchanged (i.e. the mutations V93Q, D141A, E143A, Y409G, A485L, I521L, T541G, F545L, K592A, D614N and E664R are maintained).

[0069] In some embodiments, the nucleic acid polymerase has the sequence: [ka] (SEQ ID NO:7, where X is any amino acid).

[0070] In other embodiments, the nucleic acid polymerase has the sequence: [ka] (SEQ ID NO:8, where X is any amino acid).

[0071] SEQ ID NO:7 and SEQ ID NO:8 are derived from consensus sequences obtained after alignment of motifs C and KxY of polB family polymerases (see FIG. 15 (Supplementary FIG. 10)), where the "X" amino acids are not conserved and therefore allow for some variation. SEQ ID NO:7 contains the mutations T541G, F454L, and K592A. SEQ ID NO:8 contains the mutations T541G, F454L, and K592G.

[0072] Thus, in an embodiment, there is provided a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, the nucleic acid polymerase comprising an amino acid sequence having at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO: 1, comprising SEQ ID NO: 7 or SEQ ID NO: 8. SEQ ID NO: 7 and SEQ ID NO: 8 are located from residue 536 of SEQ ID NO: 1 to residue 598 of SEQ ID NO: 1. The nucleic acid polymerase may also comprise any mutation or pattern of mutations disclosed herein. For example, mutations V93Q, D141A, E143A, Y409G / Y409N, A485L, I521L / I521H, optionally D614N, and E664K / E664R. In certain embodiments, the polymerase comprises the mutations V93Q, D141A, E143A, Y409G, A485L, I521L, optionally D614N, and E664K / E664R. The amino acid sequence of the polymerase may comprise SEQ ID NO:7 or SEQ ID NO:8, which also comprises any of the mutations disclosed herein corresponding to positions D540, D542, K591, and / or K593 of SEQ ID NO:1. These are positions 5, 7, 56, and 58 of SEQ ID NO:7 and SEQ ID NO:8.

[0073] In another aspect, a nucleic acid polymerase is provided that is capable of producing a non-DNA nucleotide polymer from a nucleic acid template, the nucleic acid polymerase comprising an amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises an E664R mutation.

[0074] The nucleic acid polymerase may comprise an amino acid sequence having at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity with the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises an E664R mutation relative to SEQ ID NO: 1. The polymerase may comprise any other specific mutation or pattern of mutations as disclosed herein. For example, the polymerase may comprise one or more of the following mutations: V93Q, D141A, E143A, and A485L relative to SEQ ID NO: 1; one or more of the following mutations: Y409, I521, and F545 relative to SEQ ID NO: 1; and / or one or more of the following mutations: Y409G, I521L or I521H, and F545L relative to SEQ ID NO: 1. The polymerase may comprise D614 relative to SEQ ID NO: 1, such as D614N.

[0075] In another aspect, a nucleic acid polymerase capable of generating a non-DNA nucleotide polymer from a nucleic acid template is provided, the nucleic acid polymerase comprising an amino acid sequence having at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence comprising a mutation at any one, all, or any combination of positions D540, D542, K591, K593, Y663, and / or Q665 relative to SEQ ID NO: 1. Optionally, the mutation at any of positions D540, D542, K591, and / or K593 is to any less bulky residue, i.e., any residue that is less sterically blocking than the wild-type residue. Optionally, the mutation at any of positions Y663 and / or Q665 is to any positively charged residue. In some embodiments, the mutation at D540 is D540A, D540G, D540S, or D540C. In particular, the mutation can be D540A. In some embodiments, the mutation at D542 is D542A, D542G, D542S, or D542C. In some embodiments, the mutation at K591 is K591G, K591A, K591C, K591M, K591S, K591D, K591P, K591N, K591T, K591E, K591V, K591Q, K591H, K591I, or K591L. In some embodiments, the mutation in K593 is K593G, K593A, K593C, K593M, K593S, K593D, K593P, K593N, K593T, K593E, K593V, K593Q, K593H, K593I, or K593L. In some embodiments, the E663 mutation can be E663K, E663R, or E663H. In some embodiments, the E665 mutation can be E665K, E665R, or E665H. The polymerase can include any other specific mutation or pattern of mutations as disclosed herein. In particular, any mutation in T541, K592, and / or E664 disclosed herein.The polymerase may also include one or more, or all of the following mutations: V93Q, D141A, E143A, and A485L relative to SEQ ID NO: 1; one or more, or all of the following mutations: Y409, I521, and F545 relative to SEQ ID NO: 1; and / or one or more, or all of the following mutations: Y409G, I521L or I521H, and F545L relative to SEQ ID NO: 1. The polymerase may include a D614 mutation relative to SEQ ID NO: 1, such as D614N.

[0076] The polymerase of the disclosure can generate non-DNA nucleotide polymers from a nucleic acid template. The nucleic acid template can be a DNA nucleotide polymer template. Non-DNA nucleotides refer to nucleotides other than deoxyribonucleotides. The polymerase can be capable of incorporating 2'-O-methyl-RNA and (2'OMe) nucleotides and / or 2'-O-(2-methoxyethyl)-RNA (MOE) nucleotides into the polymer. The polymerase can also be capable of incorporating phosphorothioate 2'-O-2-methoxyethyl-RNA (PS-MOE) nucleotides and / or locked nucleic acid (LNA) nucleotides into the polymer.

[0077] The nucleic acid polymerase may be capable of acting on a DNA primer to synthesize a 2'OMe, MOE, PS-MOE, or LNA polymer. The nucleic acid polymerase may be capable of acting on a non-DNA primer to synthesize a 2'OMe, MOE, PS-MOE, or LNA polymer, for example, the polymerase may be capable of acting on a 2'OMe-RNA primer.

[0078] It will be appreciated that many of the polymerases of the disclosure may exhibit activity towards multiple XNAs, and thus may be capable of synthesizing polymers or oligomers that contain two types of XNAs, for example polymers that contain both 2'OMe and MOE nucleotides.

[0079] To be considered capable of a particular function, the polymerase should produce polymers at least 14 nucleotides in length, suitably at least 15 nucleotides in length; more suitably at least 40 nucleotides in length, and most suitably at least 50 nucleotides in length.

[0080] Therefore, when it is discussed that a polymerase of the present disclosure is capable of incorporating a particular type of XNA, it should be understood that the polymerase is expected to be able to consistently produce polymers or lengths of at least 40 nucleotides, suitably at least 50 nucleotides.

[0081] Suitably, the polymers produced by the polymerases disclosed herein will reflect the same four bases in their information content as conventional DNA polymers, corresponding to complementary bases in the template.

[0082] The polymerases disclosed herein, including 2M polymerase, may be capable of acting on the chemicals in the table below. [Table 1]

[0083] The nucleic acid polymerase can be a polymerase that can act on a DNA primer to synthesize XNA molecules, such as 2'OMe, MOE, PS-MOE, or LNA polymers that are complementary to single-stranded nucleic acid templates. Such polymerases include polymerases that contain the mutations Y409G, I521L, T541G, F545L, K592A, and E664K (as described for SEQ ID NO: 1) in the backbone of any polymerase from the polB family. In certain embodiments, the backbone is any polB polymerase, except viral polymerases. The backbone can be that of a polymerase from the archaeal genus Thermococcus and / or Pyrococcus. The polymerase can be a mutant of the polymerase from T.gorgonarius (Tgo) (SEQ ID NO: 1). The polymerase can be of an amino acid sequence having at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence includes the mutations Y409G, I521L, T541G, F545L, K592A, and E664K relative to the amino acid sequence of SEQ ID NO:1. In certain embodiments, a nucleic acid polymerase capable of acting on a DNA primer to synthesize a 2'OMe, MOE, PS-MOE, or LNA polymer may have an amino acid sequence having at least 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence includes the mutations V93Q, D141A, E143A, Y409G, A485L, I521L, T541G, F545L, K592A, and E664K relative to the amino acid sequence of SEQ ID NO:1.

[0084] Polymerase In principle, a polymerase of the disclosure can be made by introducing specific mutations described herein into the corresponding sites of a starting polymerase or "polymerase backbone" of the operator's choice. In this way, the activity of that starting polymerase can be modified to provide activity as described herein.

[0085] The polymerase backbone can be any member of the known polB enzyme family (including the pol delta variant that shows only 36% identity with the exemplary sequence of SEQ ID NO:1). In some cases, the polymerase backbone can be any member of the known polB enzyme family except viral polymerases. The polymerase backbone can be any member of the known polB enzyme family that has at least 36%; at least 50%; at least 60%; at least 70%; or at least 80% identity with SEQ ID NO:1. At the 80% identity level, the polB enzymes of the archaeal genera Thermococcus and / or Pyrococcus are included. In certain embodiments, the polymerase backbone has at least 90% identity with SEQ ID NO:1.

[0086] Thus, in one example, there is provided a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, the nucleic acid polymerase comprising an amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence being a polymerase from the polB family that comprises any mutation or pattern of mutations disclosed herein relative to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the sequence is wild-type apart from the identified mutations.

[0087] When using other polymerase backbones, the mutations are moved to equivalent positions, as is well known in the art. For example, referring to the exemplary polymerase 6G12, the following table shows how the mutations can be moved to alternative backbones. The table shows the Pol6G12 mutations and their structurally equivalent positions in other PolB. The mutations found in Pol6G12 are shown relative to the base sequence of wild-type Tgo. The structurally equivalent residues of other well-studied B-family polymerases are given. Residues that were not mapped to the equivalent positions are shown as ND. [Table 2]

[0088] The polymerase may be a fragment of a polymerase that retains polymerase function.

[0089] Reference sequence When referring to specific amino acid residues of the polymerase using numerical addresses, the numbering is done with reference to the authentic wild-type amino acid sequence of SEQ ID NO:1 (or the nucleic acid sequence encoding it).

[0090] This should be used to locate the residue of interest, as is well understood in the art. This is not necessarily a strict counting exercise, and care should be taken with the context. For example, if the proteins of interest are of slightly different lengths, the location of the correct residue in that sequence that corresponds to (for example) E664 may require aligning the sequences and selecting the equivalent or corresponding residue, rather than simply taking the 664th residue of the sequence of interest. This is well within the purview of one of ordinary skill in the art.

[0091] A "mutation" may refer to a substitution or a truncation or deletion of the referenced residue, motif or domain. In certain embodiments, a mutation is a substitution of one type of amino acid residue with another type of amino acid residue.

[0092] Mutations may be effected at the polypeptide level, for example by synthesis of a polypeptide having a mutant sequence, or at the nucleotide level, for example by creating a nucleic acid encoding the mutant sequence, which may then be translated to produce the mutant polypeptide. If no amino acid is specified as a replacement amino acid for a given mutation site, alanine (A) may be used as the default. Suitably, the mutations used at the particular site(s) are as described herein.

[0093] Fragments are suitably at least 10 amino acids in length, suitably at least 25 amino acids, suitably at least 50 amino acids, suitably at least 100 amino acids, or suitably a majority, i.e. 387 or more amino acids, suitably at least 500 amino acids, suitably at least 600 amino acids, suitably at least 700 amino acids, suitably the full 773 amino acids, of the Tgo or TgoT polB sequence.

[0094] Sequence variations The polymerases of the disclosure may contain sequence alterations relative to the wild-type sequence, in addition to the significant mutations described in more detail herein. In particular, the polymerases of the disclosure may contain sequence alterations at sites that do not significantly impair the function or action of the polymerases described herein.

[0095] The function of the polymerase can be readily tested by manipulating the polymerase, such as described in the Examples section, to ensure that function is not impaired or significantly altered.

[0096] Therefore, sequence alterations may be made in a polymerase molecule relative to the wild-type reference sequence, provided that the polymerase retains a function that can be readily tested as defined herein.

[0097] Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: [Table 3]

[0098] When considering what mutations, substitutions, or other such changes may be made to the wild-type sequence, retention of polymerase function is paramount. Typically, conservative amino acid substitutions will be less likely to adversely affect function. Suitably, the polymerases of the present disclosure differ from the wild-type sequence only by conservative amino acid substitutions, except as discussed.

[0099] Sequence similarity / identity Sequence comparison can be performed with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate the sequence identity between two or more sequences.

[0100] Those skilled in the art will understand how to calculate the percentage identity between two nucleic acid sequences. To calculate the percentage identity between two nucleic acid sequences, it is first necessary to prepare the alignment of the two sequences, and then calculate the value of sequence identity. The percentage identity of two sequences can be different depending on (i) the method used to align sequences, such as the Needleman-Wunsch algorithm (e.g., applied by Needle (EMBOSS) or Stretcher (EMBOSS)), the Smith-Waterman algorithm (e.g., applied by Water (EMBOSS)), or the LALIGN application (e.g., applied by Matcher (EMBOSS)); and (ii) the parameters used by the alignment method, such as local alignment vs. global alignment, the matrix used, and the parameters applied to gaps.

[0101] After alignment, there are many different ways to calculate the percentage of identity between two sequences. For example, the number of identities may be divided by (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of ungapped positions; or (iv) the number of equivalent positions excluding overhangs. It will be further understood that the percentage of identity is also strongly dependent on length. Thus, the shorter the sequence pair, the higher the sequence identity that can be expected to occur by chance.

[0102] The percentage of identity between two nucleic acid sequences can then be calculated from such an alignment as (N / T)*100, where N is the number of positions where the sequences share identical residues and T is the total number of positions being compared, including gaps but excluding overhangs.

[0103] The sequence alignment may be a pairwise sequence alignment. Suitable services include Needle (EMBOSS), Stretcher (EMBOSS), Water (EMBOSS), Matcher (EMBOSS), LALIGN, or GeneWise. In one example, the identity between two amino acid sequences can be calculated using the service Needle (EMBOSS) set to default parameters, such as matrix (BLOSUM62), gap open (10), gap extension (0.5), end gap penalty (false), end gap open (10), and end gap extension (0.5). In another example, the identity between two amino acid sequences can be calculated using the service Matcher (EMBOSS) set to default parameters, such as matrix (BLOSUM62), gap open (14), gap extension (4), alternative match (1). In one example, the identity between two nucleic acid sequences can be calculated using the service Needle (EMBOSS) set to default parameters, such as matrix (DNAfull), gap open (10), gap extension (0.5), end gap penalty (false), end gap open (10), and end gap extension (0.5). In another example, the identity between two nucleic acid sequences can be calculated using the service Matcher (EMBOSS) set to default parameters, such as matrix (DNAfull), gap open (16), gap extension (4), alternative match (1).

[0104] Suitably, identity or similarity is assessed at the amino acid level over at least 400 or 500, preferably 600, 700 or 773 amino acids to the relevant polypeptide sequence(s) disclosed herein (such as any one of SEQ ID NOs: 1-6).

[0105] Similarity or identity can be calculated by comparing the full length of the amino acid sequence of the truncated nucleic acid polymerase to the relevant portion of a reference sequence (such as any one of SEQ ID NOs: 1-6). In certain embodiments, similarity or identity is calculated taking into account the full length of the reference sequence (e.g., all 773 residues of any one of SEQ ID NOs: 1-6). In certain embodiments, sequence identity of the nucleic acids of the present disclosure is calculated as a percentage of identity over all 773 residues of any one of SEQ ID NOs: 1-6.

[0106] Suitably, similarity or identity should be considered with respect to those region or regions of the sequence known to be essential for protein function and not to non-essential adjacent sequences, this is particularly important when considering homologous sequences from distantly related organisms.

[0107] When considering conserved regions, suitably the 36% of residues common to both SEQ ID NO:1 and pol delta members of the polB enzyme family should be considered as potentially important residues that are not suitably mutated in the polypeptides of the present disclosure unless otherwise indicated. Thus, suitably, the polypeptides of the present disclosure have at least 36% identity with SEQ ID NO:1, and suitably the amino acid residues constituting said at least 36% identity include amino acid residues that correspond to amino acid residues that are identical between SEQ ID NO:1 and pol delta members of the polB enzyme family. Suitably, the polypeptides of the present disclosure have at least 36% identity with SEQ ID NO:1 and at least 36% identity with pol delta members of the polB enzyme family.

[0108] For comparison, the sequence of the human DNA polymerase delta catalytic subunit is the following sequence: [ka] (SEQ ID NO: 9).

[0109] Thus, the polymerase may comprise an amino acid sequence that has at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% similarity or identity to the amino acid sequence of SEQ ID NO: 1 and at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% similarity or identity to the amino acid sequence of SEQ ID NO: 9. The polymerase may comprise an amino acid sequence that has at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1 and at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 9.

[0110] The same considerations apply to nucleic acid nucleotide sequences.

[0111] Truncation Truncations of the entire full length polymerase enzyme of the present disclosure may be made if desired. Suitably, a full length polymerase polypeptide is used as the backbone polypeptide, such as full length Tgo polymerase 1-773 as set forth in any one of SEQ ID NOs: 1-6. If truncations are used, they should be carefully checked for activity. This can be readily done by assaying the enzyme(s) described herein.

[0112] purification The polymerases of the present disclosure are advantageously thermostable. Expressing these polymerases in conventional (non-thermostable) host strains advantageously simplifies purification. For example, when expressing the polymerases of the present disclosure in conventional non-thermostable host cells, a purity of about 90% can be obtained by simply heating the host cells to 99°C, followed by centrifugation to remove the cell debris. For example, a higher purity level can be obtained by subjecting the heat-treated soluble fraction of the host cells to ion exchange and / or heparin column purification.

[0113] Suitably, the polymerase of the disclosure is not fused to any other polypeptide. Suitably, the polymerase of the disclosure is not tagged with any further polypeptide or fusion.

[0114] Fidelity It is obviously important that sufficient fidelity is maintained for accurate generation (or renaturation) of nucleic acid polymers. Suitably, the polymerases of the present disclosure retain at least 95% fidelity. The fidelity (error threshold) can be obtained as the number of errors introduced divided by the number of nucleotides polymerized. In other words, an error rate of 1% corresponds to the introduction of one error for every 100 nucleotides polymerized. In fact, the polymerases of the present disclosure achieve fidelity much better than this. An error rate of 5% or less is considered as the minimum useful fidelity level for the polymerases of the present disclosure. Suitably, the polymerases of the present disclosure have an error rate of 4% or less; suitably 3% or less; suitably 2% or less; suitably 1% or less.

[0115] Fidelity can therefore be assessed as a collective fidelity (e.g., DNA-XNA-DNA) encompassing the two conversion events (DNA-XNA and XNA-DNA), and values ​​should be adjusted or interpreted accordingly.

[0116] Methods and Uses The polymerases disclosed herein can be used to generate XNA polymers. Thus, in embodiments, a method of making a non-DNA nucleotide polymer is provided, comprising contacting a nucleic acid template with any of the nucleic acid polymerases disclosed herein under conditions conducive to polymerization.

[0117] The non-DNA nucleotide polymer may comprise or consist of 2'OMe-RNA nucleotides and / or MOE-RNA nucleotides. As such, 2'OMe-RNA nucleotides and / or MOE-RNA nucleotides may be provided during polymerization. In an embodiment, the resulting polymer is all 2'OMe-RNA polymer. In another embodiment, the resulting polymer is all MOE-RNA polymer. In an additional embodiment, the resulting polymer comprises both 2'OMe-RNA and MOE-RNA. The polymer may comprise only 2'OMe-RNA and MOE-RNA. The polymer may be an oligonucleotide.

[0118] The non-DNA nucleotide polymer may include phosphorothioate 2'-O-2-methoxyethyl-RNA (PS-MOE) nucleotides or locked nucleic acid (LNA) nucleotides. As such, PS-MOE nucleotides and / or LNA nucleotides may be provided during polymerization.

[0119] In an embodiment, the method comprises providing 2'OMe-RNA nucleotides, MOE-RNA nucleotides, PS-MOE nucleotides, LNA nucleotides, or any combination of said nucleotides to the polymerization reaction.

[0120] The method may include providing a primer, for example a DNA or non-DNA primer. The primer may be a 2'OMe-RNA primer.

[0121] The method can be used to generate polymers that are at least 14, 15, 20, 25, 40, 50, or 70 nucleotides in length.

[0122] In another aspect, there is provided a use of any of the nucleic acid polymerases disclosed herein for the production of non-DNA nucleotide polymers. The use may be for the production of oligonucleotides. The polymer may comprise 2'OMe-RNA nucleotides, MOE-RNA nucleotides, PS-MOE nucleotides, LNA nucleotides, or any combination. The polymer may comprise 2'OMe-RNA nucleotides. The polymer may comprise MOE-RNA nucleotides. The polymer may comprise 2'OMe-RNA nucleotides and MOE-RNA nucleotides. The polymer may be an all 2'OMe-RNA polymer. The polymer may be an all MOE-RNA polymer. The polymer may comprise only 2'OMe-RNA and MOE-RNA.

[0123] In some cases, the resulting polymer can act as a catalyst. The polymer can be an endonuclease. The catalytic polymer can include 2'OMe-RNA and / or MOE-RNA. The catalytic polymer can include only 2'OMe-RNA nucleotides. The polymer includes only 2'OMe-RNA nucleotides and has endonuclease activity (2'OMezyme).

[0124] In some cases, the resulting polymer is an aptamer. The aptamer may comprise 2'OMe-RNA and / or MOE-RNA. The aptamer may comprise only 2'OMe-RNA, only MOE-RNA, or only 2'OMe-RNA and MOE-RNA.

[0125] In another embodiment, there is provided the use of a nucleic acid polymerase disclosed herein to extend a DNA primer immobilized on a substrate to synthesize a non-DNA nucleic acid molecule that is complementary to a single-stranded nucleic acid template.

[0126] product In some embodiments, catalytic oligonucleotides are provided, wherein the nucleotides comprise only 2'OMe-RNA nucleotides. The catalytic oligonucleotides may have endonuclease activity. The oligonucleotides may have the sequence of 2'OMezyme disclosed herein.

[0127] In another aspect, there is provided any aptamer as disclosed herein.

[0128] remarks All of the features described in this specification (including the accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0129] For a better understanding of the present invention and to show how embodiments thereof may be practiced, reference will now be made to examples which are not intended to limit the invention in any way. EXAMPLES

[0130] (Example) Steric exclusion is a key component of enzyme substrate specificity, including polymerases. Herein, we describe the discovery of a two-residue nascent strand stereocontrol "gate" in an archaeal DNA polymerase. Engineering the gate to reduce steric bulk in the context of the previously described RNA polymerase activity is shown to enable the synthesis of 2'-modified RNA oligomers, in particular, efficient synthesis of 2'-O-methyl-RNA (2'OMe-RNA) and 2'-O-(2-methoxyethyl)-RNA (MOE-RNA) oligomers of defined random sequences up to 750 nt.

[0131] This enabled the discovery of an RNA endonuclease catalyst composed entirely of 2'OMe-RNA ("2'OMezyme") for allele-specific cleavage of oncogenic KRAS(G12D) and β-catenin CTNNB1(S33Y) mRNAs, and the elaboration of a mixed 2'OMe- / MOE-RNA aptamer with high affinity for vascular endothelial growth factor (VEGF). Our findings make these chemistries, which are used in several approved nucleic acid drugs, accessible for enzymatic synthesis and for broader exploration of directed evolution and nanotechnology.

[0132] Example 1 - A two-residue nascent strand steric gate controls the synthesis of 2'-O-methyl and 2'-O-(2-methoxyethyl)-RNA. In the experiments discussed below, we disclose the presence of a two-residue steric gate in Tgo, a replicative DNA polymerase from the hyperthermophilic archaeon Thermococcus gorgonarius, which is involved in the synthesis of a previously engineered primer-dependent RNA polymerase activity in Tgo. 10,11 Mutating this steric gate in this context enabled highly efficient synthesis of 2'OMe-RNA and, for the first time, MOE-RNA, which also enabled the in vitro evolution of the first all-2'OMe-RNA catalyst ("2'OMezyme") for mutation-specific cleavage of two oncogenic mRNA targets and the elaboration of a mixed 2'OMe / MOE-RNA aptamer with high affinity for vascular endothelial growth factor (VEGF).

[0133] ( result) The inventors have previously characterized engineered versions of Tgo, specifically TGK and TGLLK (Tgo: V93Q, D141A, E143A, Y409G, A485L, I521L, F545L, E664K). 10、11 We observed that TGLLK-mediated 2'OMe-RNA synthesis was not possible with the more challenging DNA synthesis pathway, particularly in the in vitro selection experiments. 40It was relatively inefficient on random sequence templates. We have determined the ternary structure of the homologous DNA polymerase from T. kodakarensis KOD1 (PDB ID 5OMF). 12 , and 71° C1'-C2'-O2'-C メチル Dihedral angle-adjusted 2'-O-methyl group 14,15 Enhanced RNA-DNA duplex structure 13 Using a simple static model of 2'OMe-RNA synthesis involving a 2'-methoxyl group (gauche conformation), we sought to improve 2'OMe-RNA synthesis by quasi-rational design based on the systematic elimination of unfavorable steric contacts between the bulky 2'-methoxy substituents of the nascent 2'OMe-RNA strand and the polymerase.

[0134] This approach identified the side chains of Tgo residues D540, T541, K592, D614, and E664, which are proximal to and potentially sterically clash with the 2'-methoxy group of the 2'OMe-RNA nascent strand. These residues were targeted for site-saturation mutagenesis of the TGLLK framework and screened for 2'OMe-RNA synthesis activity (SI Figure 1). Of these, T541 was of particular interest because it makes direct contact with the 3'-terminal nucleotide of the nascent (primer) strand and its positioning is critical for catalysis, i.e., the nucleophilic attack of the nascent strand terminal 3'-OH on the α-phosphate of the incoming nucleoside triphosphate substrate. Indeed, the screen identified T541G as a mutation that increases 2'OMe-RNA synthesis activity, as well as mutations K592A and K664R, which showed only modest increases in activity. Combining the mutations revealed a striking synergistic effect of the T541G and K592A mutations on 2′OMe-RNA synthesis in light of previous TGLLK mutations ( SI Fig. 1 , Fig. 1 e).

[0135] The polymerase TGLLK:T541G, K592A (hereafter referred to as 2M) (Figure 1) was synthesized by cleaving a model DNA template (TempN) that contains all possible dinucleotide combinations. 16 (Fig. 1f) and random sequence N 402M showed a marked increase in 2'OMe-RNA synthesis activity on the template (Fig. 1g). Moreover, 2M enabled long-range 750-nt 2'OMe-RNA synthesis (Fig. 1h). This suggests that residues T541 and K592 together strongly block 2'OMe-RNA synthesis, which is alleviated by mutation to less bulky side chains (T541G, K592A) (Fig. 1d). The 2M mutations also seem to remodel the polymerase-primer binding interface to such an extent that both DNA synthesis and to an even greater extent 2'OMe-RNA synthesis are unfavorable from DNA primers compared to 2'OMe-RNA primers (SI Fig. 1). Nevertheless, these mutations do not appear to impede nucleobase discrimination, as fidelity measurements suggest that the error rate of 2M synthesizing 2'OMe-RNA is in the same range as the parent polymerases TGLLK and TGK synthesizing 2'OMe-RNA and RNA, respectively (SI Table 4).

[0136] Low efficiency of XNA synthesis and reverse transcription from random templates can lead to synthesis bias and undersampling of sequence space, with a concomitant loss of library diversity and suboptimal results in repertoire selection experiments. We have demonstrated that the recently described more efficient 2'OMe-RNA reverse transcriptase C8 17 We reasoned that improving the efficiency of 2'OMe-RNA synthesis (along with the addition of 2'OMe-RNA catalysis) may enable successful in vitro evolution experiments that have been previously elusive. To this end, we pursued a novel selection of a full 2'OMe-RNA catalyst (hereafter referred to as 2'OMezyme), which to our knowledge has not been reported before. We synthesized full 2'OMe-RNAs of random sequence to which an RNA substrate is covalently attached for cleavage in cis (N 40 )repertoire 18Starting directly from R15 / 5-KRAS, we aimed to discover an endonuclease 2'O Mezyme that targets the KRAS oncogene mRNA. After 15 rounds, the selection pool was deep sequenced and screened for RNA endonuclease activity, and the most enriched active sequences were subjected to five further rounds of catalytic "maturation" selection from a doped sequence library (70% correct bases, 10% each alternative base). The most enriched 2'O Mezyme sequence R15 / 5-KRAS (hereafter referred to as R15 / 5-K) (Figure 2a) was prepared by solid-phase synthesis for further characterization.

[0137] R15 / 5-K is a highly sequence-specific RNA endonuclease that catalyzes the cleavage of its cognate substrate, KRAS G12D (cG35A) RNA, in a bimolecular reaction (25 mM Mg 2+ Inside k cat = 0.24 hours -1 ±0.05, pH 8.5, 37 °C) ( Figure 2 c), allowing multiple turnover catalysis ( SI Figure 2 ).

[0138] Cleavage was specific for the G12D (c.35G>A) mutation and essentially did not cleave "wild-type" (wt) KRAS RNA, which differs by only one nucleotide (G35) (Figure 2c). Moreover, unlike comparable variants of the standard 10-23 DNAzyme targeting the same KRAS sequence motif, R15 / 5-K was able to penetrate and cleave the long, 2.1 kb KRAS transcript while retaining specificity for the G12D mutation (c.35G>A) as well as short model RNA substrates (Figure 2e), and barely cleaved wt KRAS transcripts or transcripts with a similar nearby oncogenic mutation (G13D (c.38G>A)).

[0139] As previously observed for RNA endonuclease DNAzymes and XNAzymes (and some ribozymes), cleavage proceeds via a transesterification reaction and a 2',3'-cyclic phosphate (>p) intermediate, as indicated by MALDI-ToF mass spectrometry and electrophoretic mobility shift (EMSA) analysis of the cleavage products (SI Figure 3). However, RNA endonuclease DNAzymes and XNAzymes require divalent cations (typically Mg) for both folding and catalysis. 2+ ) and therefore has a significantly reduced catalytic activity under physiological conditions, R15 / 5-K 2'O Mezyme is a sub-physiological, low-Mg enzyme over a wide pH range. 2+ Regime (0.5-1mM Mg 2+ ) retained 70–80% of its activity (Fig. 2c) (SI Fig. 2). In fact, compared to the optimal conditions, the single turnover rate was only reduced by about 50% (1 mM Mg 2+ Inside k cat = 0.11 hours -1 ±0.01, pH 7.4, 37 °C) (Fig. 2c). Furthermore, unlike the 10-23 DNAzyme, the RNA cleavage activity of R15 / 5-K was significantly increased by Mg, even at a very low rate. 2+ It was observed even in the absence of cat = 0.001 hours -1 ±0.0002, pH 7.4, 37 °C) (SI Fig. 2). Finally, as expected for an all-2'OMe-RNA structure, R15 / 5-K demonstrated high biological stability with no significant degradation (or loss of activity) after incubation in human serum at 37 °C for 120 h (SI Fig. 4).

[0140] The potential for modularity, i.e., programmability of RNA target specificity via binding arms, is an attractive feature of some nucleic acid catalysts, such as DNAzymes,10-23, but is not shared by all. We next investigated whether the R15 / 5-K 2'O Mezyme could be retargeted to alternative mRNA substrates. Based on the predicted secondary structure of R15 / 5-K (Fig. 2a), we reprogrammed nucleotides 1-7, 39-40, and 45-51 flanking the central hairpin motif and paired them with the β-catenin (CTNNB1) proto-oncogene mRNA (c.85-111). Although the resulting 2'O Mezyme R15 / 5-CTNNB1 was weakly active, an improved mutant (R15 / 5-CTNNB1:A39G, U45A, hereafter referred to as R15 / 5-C) (Fig. 2b) was readily discovered by screening mutations in the residues adjacent to the recognition element (positions 9, 39, 42 & 45) (SI Fig. 5). The improved 2'O Mezyme R15 / 5-C was highly specific and was able to cleave only the oncogenic S33Y CTNNB1 (c.G99A) RNA substrate (Fig. 2d). It retained its specificity while retaining the capacity for multi-turnover catalysis (SI Fig. 2) and invasion of the long (4 kb) structure of the complete β-catenin transcript (Fig. 2f). The turnover rate of R15 / 5-C was significantly increased under optimal conditions (25 mM Mg 2+ Inside k cat = 0.14 hours -1 The retargeting was approximately 40% lower than that of the parent R15 / 5-K under sub-physiological low Mg conditions (pH 8.5, 37°C). 2+ Conditions (1mM Mg 2+ Inside k cat = 0.10 hours -1 ±0.01, pH 7.4, 37°C) did not affect the ratio (Figure 2d).

[0141] Next, we wondered whether 2M polymerase might be able to tackle the more challenging 2'-modified RNA substrates. Of these, the 2'-O-(2-methoxyethyl) (MOE) modification (Figure 3a) is of particular interest due to the superior biophysical and pharmacological properties of MOE-modified nucleic acids. In both 2'OMe-RNA and MOE-RNA, the 2'-substituent prefers the C3'-endo sugar conformation of the ribofuranose ring (analogous to the ribose sugar puckering in RNA (A-form)) (Figure 3b). The MOE ethylene glycol monomethyl ether modification is favored in an additional gauche orientation along O2-CCO (Figure 3c), extending the gauche effect from O4-C1-C2-O2, thereby promoting the rotational equilibrium to the C3'-endo (Figure 3b). 19 This structural preorganization (and rigidity of the MOE-RNA structure) enhances base pairing and stacking interactions with the target RNA, resulting in high antisense binding affinity of 2'OMe-RNA and MOE-RNA for RNA. Indeed, any MOE modification of a DNA oligo increases the T of the oligo bound to the complementary RNA. m will increase by 0.9 to 1.2 degrees Celsius 19 .

[0142] In addition, the gauche-oriented MOE moiety places additional hydrogen bond acceptors in the minor groove, which favors the formation of a hydrogen bond network. Thus, the MOE modification stabilizes up to three water molecules trapped between the MOE moiety and the phosphodiester backbone. 20 This hydrated “spine”, together with the steric hindrance introduced by the 2′-O-(2-methoxyethyl) group in the minor groove, provides shielding of the 5′-3′ phosphodiester bond, conferring superior biological stability and in vivo half-life to MOE-RNA. 1 Excessive hydration increases the paracellular absorption and intestinal uptake rates of MOE-modified oligonucleotides compared to unmodified oligonucleotides. 21 .

[0143] However, solution-state NMR 22 and X-ray crystallography 20The structure shows that the MOE-RNA helix adopts the previously mentioned gauche conformation for enzymatic synthesis with the bulky methoxyethyl groups protruding from the helical envelope, a challenging conformational envelope (Figure 3c). Nevertheless, we undertook the chemical synthesis of MOE-NTPs to explore the enzymatic synthesis of MOE-RNA.

[0144] Synthesis of MOE nucleosides 23 and their phosphoramidites 24 Although the MOE oligonucleotides have been established and commercial synthesis is possible, 2'-O-(2-methoxyethyl) nucleoside triphosphates (MOE-NTPs) are not commercially available and their synthesis has not been established. Therefore, we first started with commercially available 2'-O-(2-methoxyethyl) ribonucleosides and synthesized them using the established Ludwig method. 25、26 We developed a synthetic route to four MOE-NTPs by triphosphorylation based on ( SI Fig. 6 ; SI Materials & Methods ).

[0145] All four types of MOE-NTPs (MOE-ATP, MOE-GTP, MOE-CTP, MOE-m 5 We then proceeded to test the ability of the newly engineered polymerase 2M to synthesize MOE-RNA oligomers. Unlike the previous version of TGLLK, 2M (SI Fig. 7) synthesizes a model DNA template (+72 nt) and a random N 40 We were able to efficiently synthesize MOE-RNA on both library templates, enabling the synthesis of long-range MOE-RNA of 750-nt oligomers (Fig. 3def, SI Fig. 7). Incorporation of the bulkier methoxyethyl substituents at full substitution considerably shifted the electrophoretic mobility of MOE oligomers compared with DNA or 2'OMe-RNA oligomers of the same length and sequence (SI Fig. 8).

[0146] MOE is an attractive medicinal chemical modification of RNA, 2'F-DNA or 2'OMe-RNA aptamers to modulate their pharmacological properties and / or enhance their potency. Indeed, MOE-RNA and 2'OMe-RNA have similar conformational and helical preferences, as well as similar base-pairing strengths. 22、27 On the other hand, the 2'-O-(2-methoxyethyl) group has a significantly larger steric envelope (Figure 3c), which may result in steric clashes with other groups in tightly folded structures. Nevertheless, it appeared that functional mixed 2'OMe / MOE-RNA aptamers could be elaborated from all previously described 2'OMe-RNA leads. To test this, we used a well-characterized all-2'OMe-RNA aptamer for vascular endothelial growth factor (VEGF) 6 We investigated the conversion of VEGF to all-MOE-RNA or mixed 2'OMe / MOE-RNA aptamers and tested their binding activity by surface plasmon resonance (SPR). SPR revealed that the aptamer in which two of the four 2'OMe nucleotides were replaced by MOE nucleotides showed virtually identical affinity for VEGF compared to the all-2'OMe-RNA aptamer, whereas the aptamer in which three of the 2'OMe nucleotides were replaced by MOE nucleotides still bound VEGF, albeit with reduced affinity (Fig. 4; SI Table 3).

[0147] The full MOE aptamer is probably partially MOE-m 5 The use of UTP appears to have almost completely lost binding activity (SI Fig. 9), whereas the original VEGF aptamer was evolved using 2'OMe-U. Indeed, we replaced 2'OMe-U in the original aptamer with 2'OMe-m 5 Substitution with U reduced its binding affinity (SI Figure 9). The 2'OMe / MOE-RNA aptamer described here is the first mixed-chemistry aptamer developed with such a scaffold, suggesting that MOE-modified nucleic acids can fold into tight three-dimensional structures with high affinity for protein targets.

[0148] ( Consideration) Steric exclusion is a general determinant of enzyme and especially polymerase specificity. This includes the "steric gate" residues found in the active sites of most DNA polymerases that are thought to have evolved to exclude ribonucleoside triphosphates (present in much higher concentrations inside cells) from the polymerase active site in order to limit RNA incorporation into the genome. Kool et al. showed that this may be a general mechanism for steric control of nucleobase pair dimensions in the active site as a key component of the fidelity mechanism of replicative polymerases. 28 Steric factors can affect the mismatches either through direct collisions with the nascent polymerase interface or by altering the conformational equilibrium of the nascent duplex. 29 or post-synthetic inhibition of nascent strand elongation upon incorporation of noncognate nucleotides. 30 Finally, relaxation of stereocontrol is a clever strategy for engineering polymerases; for example, 9°N DNA polymerase mutants have been engineered for the incorporation of bulky 3′-substituents in Illumina next-generation sequencing. 31 or DNA polymerases are engineered for RNA synthesis or reverse transcription. 11、17 .

[0149] We previously reported that in the polB family polymerase from T. gornarius, a steric gating mutation (Y409G) as well as an efficient RNA synthesis mutation (E664K) were induced. 11 and incorporation of non-cognate 2'-5' linkages (I521L, F545L). 10We found a key mutation that enabled the synthesis of 2'OMe-RNA. The latter polymerase mutant, named TGLLK, showed increased but still inefficient synthesis of 2'OMe-RNA, suggesting that aspects of the polymerase structure were not yet fully adapted for 2'OMe-RNA synthesis. Since RNA and 2'OMe-RNA have very similar conformational preferences, we suspected a steric factor. Indeed, a systematic evaluation of potential steric clashes between the polymerase and the 2'-methoxy group of the nascent strand identified a two-residue steric gate, and their mutation to less bulky side chains (T541G, K592A) led to a dramatic increase in the efficiency of 2'OMe-RNA synthesis (Figure 1), despite the rather large steric envelope of the 2'-O-(2-methoxyethyl) group of MOE-RNA, enabling efficient MOE-RNA synthesis for the first time (Figure 3), with full-length defined or random sequence (N40) products synthesized in less than 30 min (2'OMe-RNA, less than 10 min) (SI Figure 7). Incorporation of T541G and K592A into TGLLK reduced the N-terminus as measured by densitometry. 40 The synthesis yield increased from 1% to 90% (2′OMe-RNA) and from 0% to 65% (MOE-RNA, Figures 1 g and 3 e, SI Figure 17 ).

[0150] Both T541 and K592 are part of motifs that are very highly conserved at both the sequence and structural levels in polB polymerases of archaeal, eukaryotic and even viral origin (motif C, respectively). 32 and KxY 33 ) (Fig. 5, SI Fig. 10). 34 These motifs are thought to be part of the minor groove interacting motifs involved in mismatch sensing. 35 Previous mutations to bulky, hydrophobic side chains have been shown to enhance mismatch discrimination. 36 Nevertheless, the fidelity of 2'OMe-RNA synthesis was found to be essentially unaffected compared to the parental polymerases TGK and TGLLK, which lack these mutations (SI Table 4). 10,11 The fidelity of MOE synthesis is limited by the low efficiency of currently available MOE-RNA RTs.17 Although difficult to measure due to the lack of a specific cleavage site, dropout assays suggest specific processing of the correct MOE-NTP (SI Fig. 11).

[0151] Ternary complex structure of the closely related KOD polymerase 12 According to the study, both T541 and K592 are involved in H-bond interactions with the 3'-end (T541, by water) and +1 (K592) nucleobases of the nascent strand, preventing the transfer of the 2' modification (Fig. 5b). The positive epistasis of the two mutations is consistent with the structural considerations. Both mutations are necessary to relieve the steric block, resulting in a large free volume in this critical region close to the catalytic site, and also in a nascent strand that is large enough to accommodate the 2'-O-methyl group of 2'OMe-RNA (Fig. 1) and the bulky 2'-O-(2-methoxyethyl) group of MOE-RNA (Fig. 3).

[0152] The structural model predicts that this two-residue steric gate at T541 and K592 primarily enhances the efficiency of primer 3'-end extension, rather than the nucleotide incorporation step of the polymerase catalytic cycle. Indeed, the steady-state kinetic parameters of 2M single-base incorporation for ATP (from 2'OMe-RNA primer) (SI Fig. 12) are nearly identical to those of the parent polymerase TGK (from RNA primer). On the other hand, the V for incorporation of ATP, 2'OMe-ATP, and MOE-ATP are max / k cat The values ​​are essentially identical, 2M being the K for ATP (SI Figure 12) and the parent polymerase TGK ( M = 13.3 μM) compared with the K M The results showed that the steric gate improves the fit and positioning of the 2'-modified nucleotide triphosphate into the polymerase active site, but does not accelerate the catalytic step.

[0153] Although enzymatic MOE-RNA synthesis by a polymerase has not been reported previously, a number of alternative engineering approaches for 2'OMe-RNA synthesis have been explored, including mutants of the closely related polB family KOD polymerase (KOD:N210D / Y409G / A485L / D614N / E664K). 9 2'OMe-RNA synthesis by 2M is more efficient (SI Fig. 14) and has higher fidelity (SI Table 4) (Mn 2+ The DGLNK mutations are an interesting alternative nonsteric strategy to enhance XNA-RNA synthesis, while the 2M (Y409G active site steric gate, E664K thumb subdomain mutation, and A485L "therminator" mutation) are not required forcing conditions such as ions. 37 Starting from the same (or very similar) mutational background as Tgo:E664K, which contains a negative charge proximal to the phosphodiester backbone of the nascent strand, as well as a mutation (N210D) that inactivates the 3'-5' exonuclease domain, DGLNK also contains the key D614N mutation in the thumb subdomain that removes the negative charge proximal to the phosphodiester backbone of the nascent strand. This is very similar to the previously described Tgo:E664K mutation that was found to allow efficient RNA synthesis by enlarging the positively charged polymerase interaction surface and increasing affinity for primer-template duplexes. Although not demonstrated in DGLNK, the D614N mutation, which further reduces the negative charge potential at the polymerase-nascent strand interface, may also increase the affinity of the polymerase for primer-template duplexes. At the same time, although our original model identified D614 as potentially sterically clashing with the nascent strand methoxy group, our screen did not identify a strong positive effect on 2'OMe-RNA synthesis as an isolated mutation. Nevertheless, when we reexamined the D614N mutation (2MN; 2M:D614N) in the context of 2M, we found a modest enhancement of 2′OMe-RNA and a lower degree of MOE-RNA synthesis by 2MN ( SI Fig. 14 ).

[0154] We also evaluated two other previously published polymerases, the T7 RNA polymerase mutant RGVG-M6 (T7: P266L, S430P, N433T, E593G, S633P, Y639V, V685A, H784G, F849I, F880Y) and the Taq polymerase Stoffel fragment mutant SFM4-6 (Taq SF: I614E, E615G, D655N, L657M, E681K, E742N, M747R), which have been reported to have 2'OMe-RNA synthesis activity. However, compared to 2M, the 2'OMe-RNA synthesis activity was moderate in both cases and seemed to be dependent on forcing conditions such as the presence of high concentrations of Mn2+ ions (SI Figure 13).

[0155] Finally, because in our initial screen, TGLLK: T541G, K664R (SI Fig. 1) also showed a (smaller) increase in 2'OMe-RNA synthesis efficiency compared to the single mutant T541G, we introduced K664R into the 2M polymerase, generating TGLLK: T541G, K592A, K664R (hereafter named 3M). However, polymerases 2M and 3M showed virtually identical synthetic activity, full-length yield, and stalling pattern (SI Fig. 15).

[0156] Discovery of a more efficient 2'OMe-RNA RT 17 Together, 2M opened the door to more ambitious in vitro evolution experiments, including the discovery of the first 2'OMezymes. Unlike 2'OMe-RNA aptamers, 2'OMezymes had not been reported previously, likely due to the fact that catalysts generally seem to be more sparsely distributed in nucleic acid sequence space. 38The RNA endonucleases 2'OMezyme R15 / 5-K and 2'OMezyme R15 / 5-C characterized herein differ in interesting ways from other reported RNA endonuclease DNAzymes and XNAzymes. Although highly specific, the maximum catalytic turnover is moderate, possibly due to excessively tight binding of the RNA substrate by 2'OMe-RNA, resulting in a high proportion of 2'OMezyme trapped in a conformation that is not due to product inhibition and / or catalytic activity. However, unlike, for example, standard 10-23 DNAzymes, or some XNAzymes, 2'OMezymes can be efficiently degraded at physiologically relevant low Mg concentrations. 2+ It retains most of its catalytic activity at high concentrations. This suggests that, unlike the above, 2'O Mezyme is unlikely to require a metalloenzyme, but may instead rely on acid-base catalysis similar to classical hairpin ribozymes (Hpz). Interestingly, 2'O Mezyme shares some notable secondary structure and sequence segment similarities with hairpin ribozymes, despite the lack of sequence homology. 39 (However, the hairpin and cleavage sites are reversed) (SI Fig. 16). Like Hpz, 2'O Mezyme also has the ability to catalyze RNA ligation at low temperatures (SI Fig. 16) and is capable of catalyzing RNA ligation in the presence of Mg 2+ They show activity in the absence of HPz ( SI Fig. 2 ). Consistent with this, mutations that increase sequence identity with HPz are mostly harmless ( SI Fig. 16 ).

[0157] Due to its unique structural and pharmacological properties and exceptional biological stability, 2M polymerase enables the first template-based enzymatic synthesis of MOE-RNA, a nucleic acid modification of high interest for nucleic acid therapeutics, and is facilitating its application in FDA-approved ASO drugs. 2 This makes MOE a desirable drug discovery chemistry modification of existing 2'OMe-RNA aptamers. Anti-VEGF 2'OMe-RNA aptamers 6In the case of , chimeric versions in which two or three of the 2′OMe nucleotides were replaced by MOE nucleotides could be readily elaborated and gave identical or slightly reduced binding affinity for VEGF, respectively ( Fig. 4 ), whereas complete replacement of 2′OMe-RNA with MOE RNA abolished the binding activity of this aptamer ( SI Fig. 9 ).

[0158] In conclusion, our work highlights the importance of stereocontrol in polymerase substrate specificity. The discovery of a new two-residue nascent strand steric gate complements classical active site steric gates by excluding 2'-modified nucleic acids from incorporation into the nascent strand, allowing the enzymatic synthesis of nucleic acid oligomers with bulky 2'-substituents. This enabled the efficient synthesis and evolution of 2'OMezymes as well as the elaboration of MOE-RNA synthesis and mixed 2'OMe- / MOE-RNA aptamers. Various applications are anticipated, including stereospecific synthesis of phosphorothioate (αPS)-MOE-RNA oligomers, and ASO sequences and chemistries for rapid iteration and potency enhancement of mutant aptamers.

[0159] ( Materials & Methods) (Nucleotides and Oligonucleotides) Triphosphates of 2'OMe-RNA (2'OMe-NTPs; 2'OMe-ATP, 2'OMe-CTP, 2'OMe-GTP, 2'OMe-UTP) were obtained from Jena Biosciences (Germany) and DNA (Illustra dNTPs) from GE Life Sciences (USA). Oligonucleotides were synthesized by Integrated DNA Technologies (Belgium) or Merck / MilliporeSigma (Germany). The gBlock encoding SFM4-6 was synthesized by Integrated DNA Technologies (Belgium) and gene synthesis of pET28a(+)-His6-RGVG-M6 was performed by GenScript Biotech (UK).

[0160] (Synthesis of 2'-O-MOE-NTPs) (1. General synthetic information for 2'-O-MOE-NTP synthesis) All reagents and solvents were purchased from commercial sources and used as received. Moisture-sensitive reactions were carried out in vacuum-dried glassware under a nitrogen atmosphere. 1 H, 13 C, and 31 P NMR spectra were recorded on a Bruker Avance 300, 500, or 600 MHz spectrometer using tetramethylsilane as an internal standard or a residual solvent signal [DO (d = 4.79 ppm 1 H NMR). Coupling constants are reported in Hertz (Hz) and were taken directly from the spectra. NMR splitting patterns are designated as s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). High-resolution mass spectra (HRMS) were acquired on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Samples were injected at 3 μL / min and spectra were acquired in negative ionization mode with a resolution of 15000 FWHM using leucine enkephalin as the lock mass. Precoated aluminum sheets (254 nm) were used for thin-layer chromatography (TLC). The product was purified by preparative HPLC ion exchange chromatography (SOURCE 15Q) using 0.1M / 1M TEAB buffer as eluent, followed by preparative ion-pair reversed-phase HPLC (Phenomenex Gemini 110A, C18, 10 μm, 21.2 mm×250 mm) using 1:1 (v / v) acetonitrile / 0.1M TEAB buffer / 0.05M TEAB in water as the elution system.

[0161] 2. General Procedure for Converting Triethylammonium Salts to Sodium Salts Triethylammonium nucleoside triphosphates (4–7 mg) were lyophilized in plastic tubes. The compounds were dissolved in methanol (500 μL) and NaClO4 (0.1 M in acetone, 3 mL) was quickly added. This precipitated the nucleoside triphosphate sodium salts. The tubes were centrifuged and the supernatant was discarded. The pellets were washed twice with acetone and then dried under vacuum.

[0162] (3. 2'-O-MOE-ATP, Na + Salt (2a) [ka] All solid reagents were weighed and dried in a desiccator under vacuum in a reaction flask overnight. Under nitrogen atmosphere, 2'-O-(2-methoxyethyl)adenosine (50 mg, 0.15 mmol, 1.0 equiv) and proton sponge (66 mg, 0.30 mmol, 2.0 equiv) were dissolved in trimethyl phosphate (4 mL). At -15°C, phosphoryl oxychloride (22 μL, 0.24 mmol, 1.5 equiv) was added and the reaction mixture was stirred at -15°C for 2 h. After reaction monitoring by analytical anion exchange HPLC, the reaction mixture was allowed to warm to room temperature. Tris(tetrabutylammonium) hydrogen pyrophosphate (554 mg, 0.62 mmol, 4.0 equiv) and tributylamine (370 μL, 1.60 mmol, 10.0 equiv) were dissolved in DMF (1 mL) and this solution was added to the reaction mixture. The mixture was stirred at room temperature for 30 min. Triethylammonium bicarbonate (TEAB) buffer (1 M, 20 mL) was added to quench the reaction, and the reaction mixture was extracted with diisopropyl ether (20 mL). The aqueous phase was lyophilized. The reaction mixture was purified by preparative anion-exchange HPLC with a gradient of 0.1 M TEAB to 1 M TEAB, followed by ion-pair reversed-phase HPLC with a gradient of 0.1 M TEAB to 0.05 M TEAB in 1:1 (v / v) acetonitrile / water. The product was obtained as the triethylammonium salt (31.0 mg, 20.8%) as a white powder. For analytical purposes, the triethylammonium salt was converted to the sodium salt. [ka] ESI-MS calculated [MH]: m / z = 564.03032; observed [MH]: m / z = 564.0279 (10%).

[0163] (4. 2'-O-MOE-m 5 UTP, Na + Salt (2b) [ka] All solid reagents were weighed and dried overnight in a reaction flask under vacuum in a desiccator. Under nitrogen atmosphere, 2'-O-(2-methoxyethyl)-5-methyluridine (50 mg, 0.16 mmol, 1.0 equiv.) and proton sponge (68 mg, 0.32 mmol, 2.0 equiv.) were dissolved in trimethyl phosphate (4 mL). At -15°C, phosphoryl oxychloride (22 μL, 0.24 mmol, 1.5 equiv.) was added and the reaction mixture was stirred at -15°C for 2 h. After reaction monitoring by analytical anion exchange HPLC, the reaction mixture was allowed to warm to room temperature. Tris(tetrabutylammonium) hydrogen pyrophosphate (571 mg, 0.63 mmol, 4.0 equiv.) and tributylamine (376 μL, 1.58 mmol, 10.0 equiv.) were dissolved in DMF (1 mL) and this solution was added to the reaction mixture. The mixture was stirred at room temperature for 30 min. The reaction was quenched by the addition of triethylammonium bicarbonate (TEAB) buffer (1 M, 20 mL) and the reaction mixture was extracted with diisopropyl ether (20 mL). The aqueous phase was lyophilized. The reaction mixture was purified by preparative anion-exchange HPLC with a gradient of 0.1 M TEAB to 1 M TEAB, followed by ion-pair reversed-phase HPLC with a gradient of 0.1 M TEAB to 0.05 M TEAB in 1:1 (v / v) acetonitrile / water. The product was obtained as the triethylammonium salt (42.5 mg, 28.0%) as a white powder. For analytical purposes, the triethylammonium salt was converted to the sodium salt. [ka] ESI-MS calculated [MH]: m / z = 555.01875; observed [MH]: m / z = 555.0176 (10%).

[0164] (5. 2'-O-MOE-GTP, Na + Salt (2c) [ka] All solid reagents were weighed and dried overnight in a reaction flask under vacuum in a desiccator. Under nitrogen atmosphere, 2'-O-(2-methoxyethyl)guanosine (50 mg, 0.15 mmol, 1.0 equiv.) and proton sponge (63 mg, 0.29 mmol, 2.0 equiv.) were dissolved in trimethyl phosphate (4 mL). At -15°C, phosphoryl oxychloride (21 μL, 0.22 mmol, 1.5 equiv.) was added and the reaction mixture was stirred at -15°C for 2 h. After reaction monitoring by analytical anion exchange HPLC, more phosphoryl oxychloride (21 μL, 0.22 mmol, 1.5 equiv.) was added and the reaction mixture was stirred at -15°C for another 2 h. This was repeated once more with a third addition of phosphoryl oxychloride (21 μL, 0.22 mmol, 1.5 equiv.). After reaction monitoring by analytical anion exchange HPLC, the reaction mixture was allowed to warm to room temperature. Tris(tetrabutylammonium) hydrogen pyrophosphate (1058 mg, 1.18 mmol, 8.0 equiv.) and tributylamine (696 μL, 2.92 mmol, 20.0 equiv.) were dissolved in DMF (2 mL) and this solution was added to the reaction mixture. The mixture was stirred at room temperature for 30 min. Triethylammonium bicarbonate (TEAB) buffer (1 M, 20 mL) was added to quench the reaction, and the reaction mixture was extracted with diisopropyl ether (20 mL). The aqueous phase was lyophilized. The reaction mixture was purified by preparative anion-exchange HPLC with a 0.1 M TEAB to 1 M TEAB gradient, followed by ion-pair reversed-phase HPLC with a 0.1 M TEAB to 0.05 M TEAB gradient in 1:1 (v / v) acetonitrile / water. The product was obtained as the triethylammonium salt (24.5 mg, 17.0%) as a white powder. For analytical purposes, the triethylammonium salt was converted to the sodium salt. [ka] ESI-MS calculated [MH]: m / z = 580.02523; observed [MH]: m / z = 580.0270 (11%).

[0165] (6. 2'-O-MOE-CTP, Na + Salt (2d) [ka] All solid reagents were weighed and dried overnight in a reaction flask under vacuum in a desiccator. Under nitrogen atmosphere, 2'-O-(2-methoxyethyl)cytidine (50 mg, 0.17 mmol, 1.0 equiv.) and proton sponge (71 mg, 0.33 mmol, 2.0 equiv.) were dissolved in trimethyl phosphate (4 mL). At -15°C, phosphoryl oxychloride (23 μL, 0.25 mmol, 1.5 equiv.) was added and the reaction mixture was stirred at -15°C for 2 h. After reaction monitoring by analytical anion exchange HPLC, more phosphoryl oxychloride (23 μL, 0.25 mmol, 1.5 equiv.) was added and the reaction mixture was stirred at -15°C for another 2 h. After reaction monitoring by analytical anion exchange HPLC, the reaction mixture was allowed to warm to room temperature. Tris(tetrabutylammonium) hydrogen pyrophosphate (1198 mg, 1.32 mmol, 8.0 equiv.) and tributylamine (788 μL, 3.32 mmol, 20.0 equiv.) were dissolved in DMF (2 mL) and this solution was added to the reaction mixture. The mixture was stirred at room temperature for 30 min. Triethylammonium bicarbonate (TEAB) buffer (1 M, 20 mL) was added to quench the reaction, and the reaction mixture was extracted with diisopropyl ether (20 mL). The aqueous phase was lyophilized. The reaction mixture was purified by preparative anion-exchange HPLC with a 0.1 M TEAB to 1 M TEAB gradient, followed by ion-pair reversed-phase HPLC with a 0.1 M TEAB to 0.05 M TEAB gradient in 1:1 (v / v) acetonitrile / water. The product was obtained as the triethylammonium salt (20.0 mg, 12.7%) as a white powder. For analytical purposes, the triethylammonium salt was converted to the sodium salt. [ka] ESI-MS calculated [MH]-: m / z = 540.01908; observed [MH]-: m / z = 540.0197 (65%) (reported as TEA salt).

[0166] (Rational selection of polymerase models and mutagenesis sites) Minilibraries for the introduction of single mutations at specific polymerase residues were constructed using the ternary crystal structure of a DNA primer-template duplex in complex with the closed Thermococcus kodakarensis KOD1 DNA polymerase, a close B family homologue of the Thermococcus gorgonarius polymerase mutant used in this study, and dATP entering the active site (PDB ID 5OMF)1. The crystal structure was loaded into Pymol, and the appropriate 2'-hydrogen atoms of the primer nucleotides were manually replaced with oxygen atoms using the "build" function in Pymol. The hydrogen atom of the newly introduced 2'-hydroxyl moiety was then similarly replaced with a methyl group. The added dihedral angle was manually adjusted to 71° (gauche conformation). 2,3 This model served as a structural guide to calculate distances from polymerase residues to the introduced 2'-O-methyl carbon atoms of the primer and to identify sites of steric clashes. These were targeted for site-saturation mutagenesis to relieve steric hindrance and increase polymerase processivity at 2'OMe-RNA.

[0167] Cloning of expression constructs and site-saturation mutagenesis Thermococcus gorgonarius (Tgo) polymerase mutant TGLLK (Tgo: V93Q, D141A, E143A, Y409G, A485L, I521L, F545L, E664K) as parental plasmid 5 The pASK75 plasmid encoding 4 Inverse PCR (iPCR) was performed with overlapping forward and reverse primers to introduce a BsaI restriction site (see Supplementary Table 1) at 3′-Gamma-1′. Cloning primers for site-saturation mutagenesis contained degenerate NNS codons (N for all bases, S for G and C) to introduce a mini-library of 32 codons encoding all 20 amino acids on a single residue (see Supplementary Table 1).

[0168] iPCR reactions were performed with 20 ng of DNA template using polymerase Q5 (New England Biolabs, NEB) and forward and reverse primers (0.5 μM each) and dNTPs (200 μM each). iPCR reactions were incubated in a thermocycler with the following program: 98°C, 30 s; (98°C, 10 s; 50–72°C, 30 s; 72°C, 3 min); 72°C, 3 min). iPCR products were purified using a PCR purification kit (Qiagen). Products were restriction cut with BsaI and DpnI (NEB) and agarose gel purified where appropriate. Products were ligated with T4 DNA ligase and purified with another cleanup kit (Bioline). Cloned constructs were transformed into chemically or electrically competent E. coli 10-β cells (NEB) or E. coli BL21 CodonPlus-RIL cells (Agilent) and plated onto TYE agar plates supplemented with the appropriate antibiotic.

[0169] (Primer extension reaction) Analytical primer extension reactions were performed in 1x Thermopol buffer (NEB) supplemented with MgSO4 (4mM). Primers (100nM) were extended with purified polymerase (10-100μg / mL) on template (200nM) using the appropriate nucleoside triphosphates (125-250μM each) in a 10μL reaction volume. Reactions were carried out at 65°C. Primer extension products were analyzed by urea-PAGE. All extensions with MOE-NTPs on defined sequence templates TempNpure required post-synthesis template capture with 10-fold excess of antisense template, Turbo DNase (Invitrogen) treatment followed by proteinase K (NEB) treatment and loading onto a urea-PAGE gel with 10-fold excess of antisense template. Primer extension with MOE-NTPs on the template sfGFP required a polymerase concentration of 500 μg / mL.

[0170] (Enzyme-Linked Oligonucleotide Assay (ELONA) Polymerase Activity Assay (PAA)) Site-saturation mutagenized polymerase mini-libraries were transformed into E. coli 10-β cells and plated on TYE agar plates supplemented with ampicillin. For all single mutant mini-libraries, 2x94 clones were hand-selected from the agar plates and used to inoculate 1 mL of 2x94 liquid starter cultures in 2xTY supplemented with ampicillin (100 μg / mL) in 96 deep-well plates (Nunc) along with two control wells per plate with the parent polymerase TGLLK. Cultures were grown overnight at 37°C. The next day, 100 μL of each culture was used to inoculate a new 1 mL culture in a new plate and grown at 37°C until the cultures reached mid-phase. Protein expression was then induced with 200 μg / L anhydrotetracycline for 2 h at 37°C. Cultures were stored overnight at 4°C. Cells were harvested by centrifugation and resuspended in 100 μL of Thermopol buffer. Cells were transferred to 200 μL 96-well plates and lysed for 30 min at 75° C. Lysed cells were cooled in an ice-water bath and the lysate was clarified by centrifugation at 4° C. The clarified lysate was transferred to a new 200 μL 96-well plate and stored at 4° C.

[0171] Primer extension reactions were carried out in 1x Thermopol buffer (NEB) supplemented with MgSO4 (4mM). Biotinylated primer FD (100nM) was extended on template TempNpure (200nM) with 2'-O-methyl ribonucleoside triphosphates (125μM each) by the polymerase mutants in whole cell lysates in a reaction volume of 10μL. Reactions were carried out at 65°C.

[0172] Biotinylated primer extension products were diluted in PBS supplemented with 0.1% (v / v) Tween 20 (PBST) and bound on streptavidin-coated plates (Roche) for 1 h at room temperature. After all incubation steps, the respective supernatants were discarded. Hybridized templates were then removed by two 1 min denaturation steps with 0.1 M NaOH. After a neutralization step with PBST, a digoxigenin-labeled oligonucleotide probe (DIGN25, 60 nM in PBST) was applied for 1 h, hybridizing only to efficiently extended primers and with increasing affinity for longer extension products.

[0173] After three washing steps with PBST, anti-digoxigenin antibody fragments conjugated to horseradish peroxidase (1:3,000 dilution in PBST, Roche) were allowed to bind on the plate for 1 h. After four PBST washes, the assay was developed by adding 3,3',5,5'-tetramethylbenzidine (TMB, 1-Step Ultra TMB-ELISA, Thermo) and incubating until the formation of a blue color was complete (as judged by TGLLK control wells). The enzymatic reaction was stopped by the addition of 1 M H2SO4, changing the color to yellow. The absorbance was read at 450 nm in a plate reader.

[0174] Screening hits were miniprepared, sequenced, and polymerase activity was verified in an extension reaction with fluorescently labeled primer FD as described above, with lysate loading adjusted by SDS-PAGE analysis and normalization based on polymerase band intensity. Primer extension products were analyzed by urea PAGE.

[0175] (Polymerase expression and purification) Expression and purification of the polymerase was carried out essentially as described above. 6Briefly, a starter culture of E. coli BL21 CodonPlus-RIL cells (Agilent) was inoculated from a single colony and grown overnight at 37°C in 2xTY medium supplemented with ampicillin (100 μg / mL) and chloramphenicol (25 μg / mL). It was used to inoculate 30 mL (small scale) or 1 L (large scale) of the same medium the following day. Cultures were grown to mid-log phase and induced with 200 μg / L anhydrotetracycline for 4 h at 37°C. After overnight storage at 4°C, harvested cells were lysed at 75°C for 30 min and lysates were clarified by centrifugation. His-tagged polymerases were benchtop purified by gravity flow over Ni-NTA agarose resin (Qiagen) and non-His-tagged polymerases were benchtop purified by gravity flow over DEAE Sepharose fast flow anion exchange resin (GE Healthcare). The eluted fractions were then loaded onto a 16 / 10 Hi-Prep Heparin FF column (Cytiva Life Sciences) and eluted with NaCl between 0.5 and 0.8 M. Appropriate fractions were dialyzed (Amicon Ultra Centrifugal Filters, Millipore) into 2x polymerase storage buffer (1 M KCl, 2 mM 290 EDTA, 20 mM Tris pH 7.4) and stored at -20°C in 50% glycerol.

[0176] (Synthesis of long fluorophore-labeled RNA) Human cDNA clones of KRAS (transcript variant b, accession number NM_004985) and CTNNB1 (transcript variant 1, accession number NM_001904) in plasmids pCMV6-XL6 (SP6 promoter) (cat. no. SC109374) and pCMV6-XL5 (T7 promoter) (cat. no. SC107921), respectively, were obtained from OriGene, USA. Site-directed mutagenesis was performed using the QuikChange II kit (Agilent Technologies, USA) according to the manufacturer's protocol. KRAS mutations G12D (c.35G>A) and G13D (c.38G>G) and CTNNB1 mutation S33Y (c.98C>A) were introduced using the primer sets shown in Supplementary Table 2 ("Quik_KRAS_G12D_Fw / Rev", "Quik_KRAS_G13D_Fw / Rev" or "Quik_CTNNB_G12D_Fw / Rev"), and the resulting plasmids were cloned and verified by Sanger sequencing (Source Biosciences, UK). Long RNA substrates equivalent to the complete KRAS and CTNNB1 mRNA transcripts with 5' fluorescein ("Sub_KRas_ORF" and "Sub_CTNNB1_ORF", respectively) were prepared using HiScribe T7 and SP6 RNA synthesis kits (NEB, USA) according to the manufacturer's protocol, with a 4:1 ratio of 5'-fluorescein ApG dinucleotide (IBA Life Sciences, Germany) to GTP, and with template plasmids linearized with XmaI (NEB, USA). Reactions were then treated with TURBO DNase (Invitrogen / Thermo Fisher Scientific, USA), and the RNA transcripts were purified using the RNeasy mini kit (Qiagen, Germany).

[0177] (2'O Mezyme selection) In brief, we synthesized chimeric RNA-2'OMe-RNA random sequence libraries using the same method as previously described for XNAzymes. 7,8The first library synthesis reaction was carried out at 50°C for 1 h and 65°C for 2 h with 1 μM RNA primer “P1_KRas12[G12D]”, 2 μM DNA template “N40libtemp_KRas12”, 1.3 μM 2M polymerase and 0.125 mM (each) 2'OMe-ATP, 2'OMe-CTP, 2'OMe-GTP and 2'OMe-UTP in Thermopol buffer (NEB, USA). The (5' biotinylated) single-stranded chimeric RNA-2'OMe-RNA library was captured using MyOne Streptavidin C1 Dynabeads (Invitrogen / Thermo Fisher Scientific, USA) and selected using a strategy similar to that described previously. 7 The (non-biotinylated) DNA template was denatured and removed using 0.1 N NaOH as in . The library was then purified by urea PAGE. The library was annealed in nuclease-free water (Qiagen, Germany) for 60 s at 80 °C and 5 min at RT, and then the selection reaction was performed by incubating in 2'O Mezyme selection buffer (30 mM EPPS pH 7.4, 150 mM KCl, 1 mM MgCl2) at 37 °C. The reaction times were changed as follows: rounds 1-11; overnight (approximately 16 h), rounds 11&12; 1 h, and rounds 13-15; 30 min.

[0178] 2'OMe-RNA reverse transcription was performed using 1 μM polymerase C8 with 0.2 μM of 5' biotinylated primer "RT_Ebo" in Thermopol buffer (NEB, USA) with an additional 2 mM MgCl2, 200 μM of each dNTP. 9PCR was performed at 65 °C for 17 h using 100% ... For the second step ("in-nest") PCR, the above cycle conditions were used with 1 μL of unpurified out-nest PCR product as template in a 50 μL reaction, using 0.5 μM forward primer "dP2_KRas12" and 0.5 μM reverse primer "RT_Ebo_in". Reactions were analyzed by electrophoresis on 4% NGQT-1000 agarose (Thistle Scientific, UK) gels with GelStar stain (Lonza, Switzerland). Bands of appropriate size were purified using a gel extraction kit (Qiagen, Germany) according to the manufacturer's instructions. Purified DNA was used as polyclonal template for either sequencing library PCR (see below) or preparative PCR ("in-nest" PCR scaled up to 500 μl) for DNA template generation for XNA synthesis. Single-stranded DNA templates were isolated using streptavidin beads and ethanol precipitated before further use.

[0179] We then performed five rounds of "maturation" selection (30 min reaction at 37 °C in 2'O Mezyme reaction buffer) using the spiked library synthesized as above with DNA template "R15_1libtemp_KRas12" based on the sequence of the most abundant clone in round 15 (containing 84,674 of 3,942,063 deep sequencing reads; approximately 2%). 2'O Mezyme "R15 / 5-K" was the most abundant clone in round 5 of the maturation selection (containing 1,291 of 5,507,023 deep sequencing reads; 0.02%).

[0180] (Deep Sequencing) Deep sequencing was previously described. 7 The sequencing was performed using the MiSeq platform (Illumina, USA) as described in. To add the necessary priming sites, the 2'O Mezyme selected pool was converted into a sequencing library by PCR using primers "P5_P2_KRas12" and "P3_RT_Ebo_in".

[0181] (Synthesis of 2'O Mezyme for characterization) For initial screening of 2'O Mezyme activity and evaluation of point mutations, 2'O Mezymes were synthesized using the RNA primer "P2_Ebo" and 3' biotinylated DNA template as shown in Supplementary Table 2 using polymerase 2M as described previously. 7 The primers were isolated using MyOne Streptavidin C1 Dynabeads (Invitrogen / Thermo Fisher Scientific, USA) as described in. Following denaturation and removal of the DNA template strand with 0.1 NaOH, the 2'O Mezyme was incubated in 0.8 N NaOH at 65°C for 1 h to completely hydrolyze the primer RNA.

[0182] 2'O Mezyme for all other characterization experiments was synthesized by solid-phase phosphoramidite chemistry by Merck / MilliporeSigma (Germany).

[0183] (2'O Mezyme reaction) RNA cleavage assays were performed in trans using PAGE purified 2'O Mezyme and RNA substrates, annealed as above, and incubated at 37°C in 2'O Mezyme selection buffer (30 mM EPPS pH 7.4, 150 mM KCl, 1 mM MgCl2) supplemented with RNasin ribonuclease inhibitor (Promega, USA) or in 30 mM EPP pH 8.5, 150 mM KCl, 25 mM MgCl2. 2+ For titration experiments, 2'O Mezyme selection buffer was further supplemented with magnesium chloride (MgCl2), for pH titration experiments, 150 mM KCl, 1 mM MgCl2 and 50 mM buffers as follows: HEPES (pH 5.0-6.0), EPPS (pH 6.5-8.75), CHES (pH 9.0-12.0) were used, for reactions without magnesium, 30 mM EPPS pH 7.4, 150 mM KCl, 5 mM EDTA were used.

[0184] Steady state before one turnover (K m / k cat The pseudo-first-order reaction rate (kob) under the conditions described above 8 As in, it was determined from three independent reactions (annealed separately) with 5 μM catalyst and 1 μM substrate, fitted using Prism9 (GraphPad Software, USA). For the multi-turnover reaction, 1 μM substrate was reacted with 10 nM 2'O Mezyme in 2'O Mezyme selection buffer at 37 °C.

[0185] For the reverse RNA ligation reaction, the products of the large-scale "Sub_KRas_12[G12D]" RNA cleavage reaction catalyzed by 2'O Mezyme "R15 / 5-K" were purified by urea PAGE and used as substrates. 5 μM of 2'O Mezyme "R15 / 5-K" and 1 μM (each) of the 5' and 3' RNA cleavage products were annealed in water as described above, then diluted in 2'O Mezyme selection buffer with or without magnesium chloride, flash frozen on dry ice, and then incubated in the reaction at -7°C or 37°C for 20 hours. The "supercooled" samples were directly incubated at -7°C without prior freezing on dry ice.

[0186] (Analysis of 2'O Mezyme-catalyzed RNA cleavage products) The substrate RNA "Sub_KRas_12[G12D]" was reacted with 2'O Mezyme "R15 / 5-K" under selected conditions, and the 5' RNA cleavage products were purified by urea PAGE. 8 The samples were analyzed by MALDI-ToF mass spectrometry using an Ultraflex III TOF-TOF instrument (Bruker Daltonik, Bremen, Germany) in positive ion mode as described above.

[0187] Enzymatic removal of 3'-terminal phosphates was assayed by urea-PAGE gel shift after incubation in calf intestinal phosphatase (CIP) (NEB, USA) or T4 polynucleotide kinase (PNK) (NEB, USA) in manufacturer's buffer for 30 min at 37° C. Hydrolysis of cyclic phosphates was achieved by incubation in 10 mM glycine pH 2.5 for 30 min at room temperature.

[0188] 2'O Mezyme Serum Stability Analysis PAGE-purified 2'O Mezyme "R15 / 5-K" and DNAzyme "1023_KRasC" were annealed in water as described above and then incubated (at 5 μM) in 95% human serum (MilliporeSigma, Germany) at 37° C. Remaining full-length catalyst was quantified on urea PAGE gels stained with SYBR Gold (ThermoFisher Scientific, USA).

[0189] (Analysis of aptamer binding by surface plasmon resonance (SPR)) 2'OMe / MOE-RNA aptamers were synthesized from the RNA primer Prim1 and the 3'-biotinylated DNA template Temp_ARC224 (Supplementary Table 1) as described in the section "Synthesis of 2'OMezyme for characterization" using 2'OMe / MOE-NTPs. 2'OMe / MOE-RNA aptamers were annealed at 1–10 μM in nuclease-free water by heating to 95 °C for 5 min and equilibrating at RT for 10 min. They were then diluted and analyzed in PBS + 0.1% (v / v) Tween 20 (PBS-Tw). Surface plasmon resonance (SPR) measurements were performed at 20 °C and a flow rate of 20 μL / min using a BIAcore 2000 instrument (GE Life Sciences, UK). The surface of a CM4 sensor chip (GE Life Sciences, UK) was coated with a Neutravidin (Pierce 31000, ThermoFisher Scientific, USA) surface (~8000 RU per flow cell) using an amine coupling kit (GE Life Sciences, UK) and run with 5 mM NaOAc (sodium acetate) pH 5.5. The chip was equilibrated with PBS Tw and left to run overnight until signal drift had settled. ~2000 RU biotinylated human VEGF165 (Bio-Techne, USA) was captured (except for reference cells) and blocked with excess free biotin. A 50 μL aptamer sample of a concentration series (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM, 15.6 nM, 7.8 nM, 3.9 nM) was injected for 150 s and dissociation was recorded for 600 s in PBS-Tw. One injection of aptamer outside the concentration series was performed at 100 nM (50 μL) in PBS-Tw. After each injection, the sensor surface was regenerated with two 5 μL injections of 10 mM NaOH + saline (137 mM NaCl, 2.7 mM KCl).

[0190] To obtain the best fit, the SPR data had to be fitted to a biexponential heterogeneous dissociation / association model and kinetic parameters had to be determined from two independent data sets per aptamer using online reference subtraction. For the ARC224 MOE-AGC aptamer, the two lowest concentration points were not included in the analysis and discarded as outliers due to insufficient binding signal. For nucleic acid-protein interactions, a heterogeneous model has been described that establishes deviations from the homogeneous 1:1 binding model and accounts for two conformationally distinct populations of DNA aptamer-bound VEGF. 10 .

[0191] The dissociation and association rate constants were obtained by fitting the observed response signal R using the following two equations:

[0192] Heterogeneous dissociation:

number

[0193] Heterogeneous Association:

number

[0194] (NGS for 2'OMe synthesis and RT fidelity analysis) For 2'OMe-RNA synthesis, ssDNA template was generated by linearization of pASK_TGO plasmid with EcoR1 followed by shrimp alkaline phosphatase treatment and restriction with BamHI. The 369 ntd dsDNA fragment was gel eluted and treated with lambda exonuclease (NEB) to generate single-stranded template for RNA / 2'OMe-RNA synthesis. 2'OMe-RNA synthesis was performed in a 20 μL reaction volume and the modFD-N25-TGO682F primer and the ssDNA template generated as described above were annealed in 1x Thermopol buffer containing 200 μM rNTPs or 200 μM 2'OMe-NTPs at 95 °C for 2 min followed by 55 °C for 5 min. RNA and 2'OMe-RNA synthesis were performed using TGK polymerase (RNA) and TGLLK or 2M or 3M (2'OMe-RNA) synthesis, respectively.

[0195] Synthetic transcripts containing 5' biotin modifications were bound to Dynabeads™ M-280 streptavidin beads (Invitrogen) and purified by stripping the template using 0.2 N NaOH. Magnetic beads immobilized with RNA or 2'OMe-RNA were used for reverse transcription with SSIII enzyme (ThermoFisherScientific). RT reactions were performed on the beads using RT_primer TagR1-N25-TGO642R with an N25 internal barcode for PCR and sequencing error correction. RT reactions were performed according to the vendor's guidelines for SSIII. cDNA bound to RNA or 2'OMe-RNA on the beads was washed twice with 1X BWBS, stripped with 0.2 N NaOH, neutralized with Tris buffer, and then used for generating sequencing libraries. RT was repeated three more times, and the eluted cDNA was used for library preparation for deep sequencing.

[0196] The cDNA (25 μL) was added to a 50 μL PCR reaction with the forward primer primer HiSeq_ModFD and the unique barcode identifier primer HiSeq_TagR1xx (Supplementary Table 5), samples were demultiplexed, and adapters for Illumina sequencing were introduced using Q5 polymerase (NEB).

[0197] Barcoded fidelity libraries were pooled and sequenced on an Illumina MiSeq for 150 cycles of PE reads. Fidelity analysis was performed using Burrows-Wheeler Aligner (BWA)11 and Samtools12 and custom scripts can be found on GitHub: https: / / github.com / holliger-lab / fidelity-analysis. Average error rates (Supplementary Table 4) and base substitutions were calculated for RNA and 2'OMe-RNA per 106 bases sequenced (Supplementary Tables 6 and 7). 9 .

[0198] (Steady-state dynamics) Steady-state kinetic parameters for NTP incorporation by 2M were determined by performing initial rate measurements of single incorporation of either ATP, 2'OMe-ATP, or MOE-ATP. To generate 2'OMe-RNA / DNA substrates, a 20-mer 2'OMe-RNA primer FD was end-labeled with 5'6-carboxyfluorescein and annealed to a 52-mer DNA template BFL770 (Supplementary Table 1) at a 1:1.2 molar ratio. Reactions were incubated in 1X Thermopol buffer, 6 mM Mg 2+ The reactions were carried out at 50 °C with NTP concentrations ranging from 0.5 to 250 μM in a mixture containing 100 nM 2'OMe-RNA / DNA. Enzyme concentrations and reaction times were selected to maintain initial velocity conditions. 25 μL reactions were stopped by adding a quenching solution containing 100 mM EDTA, 80% deionized formamide, 0.25 mg / ml bromophenol blue, and 0.25 mg / ml xylene cyanol. Additionally, less than 20% of the primer was extended for steady-state conditions, if necessary.

[0199] Products and substrates were separated on a 22% denaturing (8 M urea) polyacrylamide gel. Obtained bands were quantified using a Cytiva Typhoon RGB imager in fluorescence mode. Steady-state kinetic parameters (K M , k cat ) was determined. Data are means and standard errors from three independent experiments.

[0200] (Transcription reaction by RGVG-M6) The DNA template for the transcription reaction was generated by PCR amplification of a 901-bp region on a plasmid encoding sfGFP under the T7 promoter, using 0.5 μM forward primer “5T7.for” and 0.5 μM reverse primer “pCUN_Do.rev” and cycling conditions were 95°C for 30 s, 30× [95°C for 10 s, 69°C for 30 s, 72°C for 30 s], and 72°C for 2 min.

[0201] For highly permissive conditions, reactions contained 125 nM DNA template, 200 nM T7 RNAP WT or its mutant RGVG-M613, 1.5 mM MnCl2, 7.5 mM each NTP or 1 mM each 2'OMe-NTP, and 0.1 U yeast inorganic pyrophosphatase. To compare the yield of 2'OMe-RNA synthesis by 2M and RGVG-M6, reactions were carried out as described in 13 under equimolar nucleic acid input of 0.5 pmol primer (2M) and 0.5 pmol DNA template (50 nM, RGVG-M6), and 50 nM RGVG-M6 polymerase at a polymerase:template ratio of 1:1. Reactions were treated with turbo DNase and proteinase K, followed by denaturing PAGE.

[0202] (References) References in the Background section, explanations of Figures 1-5, and Example 1, Results and Discussion [Table 4] TIFF2024534987000025.tif183170 Description of Figures 6-19 (Supplementary Figures 1-17) and references in Materials & Methods [Table 5]

[0203] Supplementary Table 1 lists SEQ ID NOs: 45 to 87 in order. Supplementary Table 2 lists SEQ ID NOs: 88 to 127 in order. Supplementary Table 5 lists SEQ ID NOs: 128 to 142 in order.

[0204] (Supplementary table) Supplementary Table 1: Primers and templates for all polymerase studies, mutagenesis, 2'OMe-RNA and MOE-RNA synthesis, and ARC224 aptamer variant synthesis. The codons targeted for mutagenesis are highlighted in bold. The different chemistries are highlighted as follows: black=DNA, red=RNA, purple=2'OMe-RNA. [Table 6] TIFF2024534987000028.tif248170TIFF2024534987000029.tif249170TIFF2024534987000030.tif102170

[0205] (Supplementary Table 2: Primer and template sequences for 2'O Mezyme) The different chemistries are highlighted as follows: black=DNA, red=RNA, purple=2'OMe-RNA (NB - the 2'OMe-RNA oligos shown here were prepared by solid-phase synthesis rather than with a polymerase). [Table 7] TIFF2024534987000032.tif247170TIFF2024534987000033.tif249170TIFF2024534987000034.tif128170

[0206] (Supplementary Table 3: Kinetic data obtained via SPR curve fitting) Each row of fitted parameters represents one concentration series (8 individual injections of 2-fold dilution series; MOE-AGC: 6 individual injections; as described in Materials & Methods). Standard errors of the means (sem) are shown. [Table 8]

[0207] Supplementary Table 4: Barcoded next-generation sequencing (NGS) 9 Fidelity of 2'OMe-RNA synthesis (RNA synthesis for TGK) measured by [Table 9] a The fidelity of TGK for RNA synthesis is 1.03 × 10 -3 (average error rate) 15 .

[0208] Supplementary Table 5: Primer and template sequences for RNA and 2'OMe-RNA fidelity Note: The reverse primer for the sequencing library carries a six-letter barcode upstream of the NNN to demultiplex the samples for analysis. [Table 10] TIFF2024534987000038.tif175170

[0209] [Table 11]

[0210] [Table 12]

Claims

1. 1. A nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, comprising an amino acid sequence having at least 36% identity with the amino acid sequence of SEQ ID NO: 1; the amino acid sequence is mutated at T541 and / or K592 relative to the amino acid sequence of SEQ ID NO: 1; and The nucleic acid polymerase as described above, wherein the amino acid sequence is mutated at E664 relative to the amino acid sequence of SEQ ID NO:

1.

2. 2. The nucleic acid polymerase of claim 1, wherein the amino acid sequence comprises: i) a T541 mutation and a K592 mutation; ii) a T541 mutation and an E664 mutation; or iii) a T541 mutation, a K592 mutation, and an E664 mutation.

3. 3. The nucleic acid polymerase of claim 1, wherein the T541 mutation is T541G, T541S, T541A, T541C, T541D, T541P, or T541N.

4. The nucleic acid polymerase of claim 3, wherein the T541 mutation is T541G.

5. 3. The nucleic acid polymerase of claim 1, wherein the K592 mutation is K592G, K592A, K592C, K592M, K592S, K592D, K592P, K592N, K592T, K592E, K592V, K592Q, K592H, K592I, or K592L.

6. The nucleic acid polymerase of claim 5, wherein the K592 mutation is K592A or K592G.

7. 3. The nucleic acid polymerase of claim 1, wherein the E664 mutation is E664H, E664K, or E664R.

8. 3. The nucleic acid polymerase of claim 1, wherein the amino acid sequence comprises the mutations T541G and K592A.

9. The amino acid sequence i) one or more, or all, of the following mutations relative to SEQ ID NO: 1: V93Q, D141A, E143A, and A485L; and / or ii) one or more, or all, of the following mutations relative to SEQ ID NO: 1: Y409, I521, and F545; and / or iii) one or more, or all, of the following mutations relative to SEQ ID NO: 1: Y409G, I521L or I521H, and F545L; The nucleic acid polymerase according to claim 1 or 2, comprising:

10. 3. The nucleic acid polymerase of claim 1, wherein the amino acid sequence comprises a D614 mutation relative to SEQ ID NO:

1.

11. 11. The nucleic acid polymerase of claim 10, wherein the D614 mutation is D614N.

12. the amino acid sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to the amino acid sequence of SEQ ID NO: 1; and / or The amino acid sequence i) the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, wherein residues 93, 141, 143, 409, 485, 521, 541, 545, 592, and 664 are unchanged; and / or ii) the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, wherein residues 93, 141, 143, 409, 485, 521, 541, 545, 592, 614, and 664 are unchanged; and / or has at least 36%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% similarity or identity to 3. The nucleic acid polymerase of claim 1, wherein the amino acid sequence comprises SEQ ID NO: 7 or SEQ ID NO:

8.

13. 1. A nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, comprising an amino acid sequence having at least 36% identity with the amino acid sequence of SEQ ID NO: 1; The nucleic acid polymerase as described above, wherein the amino acid sequence is mutated at E664R relative to the amino acid sequence of SEQ ID NO:

1.

14. 14. The nucleic acid polymerase of claim 1 or 13, wherein the amino acid sequence comprises one or more of the following mutations relative to SEQ ID NO: 1: D540, D542, K591, K593, Y663, and Q665, or any combination thereof.

15. 1. A nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a nucleic acid template, comprising an amino acid sequence having at least 36% identity with the amino acid sequence of SEQ ID NO: 1; The nucleic acid polymerase, wherein the amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO: 1 at one, all, or any combination of positions D540, D542, K591, K593, Y663, and / or Q665 relative to SEQ ID NO:

1.

16. (i) the mutation at D540 is D540A, D540G, D540S, or D540C; and / or (ii) the mutation at D542 is D542A, D542G, D542S, or D542C; and / or (iii) the mutation at K591 is K591G, K591A, K591C, K591M, K591S, K591D, K591P, K591N, K591T, K591E, K591V, K591Q, K591H, K591I, or K591L; and / or (iv) the mutation at K593 is K593G, K593A, K593C, K593M, K593S, K593D, K593P, K593N, K593T, K593E, K593V, K593Q, K593H, K593I, or K593L; and / or (v) the mutation at Y663 is Y663K, Y663R, or Y663H; and / or (vi) the mutation at Q665 is Q665K, Q665R, or Q665H; The nucleic acid polymerase of claim 14.

17. (i) the mutation at D540 is D540A, D540G, D540S, or D540C; and / or (ii) the mutation at D542 is D542A, D542G, D542S, or D542C; and / or (iii) the mutation at K591 is K591G, K591A, K591C, K591M, K591S, K591D, K591P, K591N, K591T, K591E, K591V, K591Q, K591H, K591I, or K591L; and / or (iv) the mutation at K593 is K593G, K593A, K593C, K593M, K593S, K593D, K593P, K593N, K593T, K593E, K593V, K593Q, K593H, K593I, or K593L; and / or (v) the mutation at Y663 is Y663K, Y663R, or Y663H; and / or (vi) the mutation at Q665 is Q665K, Q665R, or Q665H; The nucleic acid polymerase of claim 15.

18. the non-DNA nucleotide polymer comprises 2'-O-methyl-RNA (2'OMe-RNA) nucleotides and / or 2'-O-(2-methoxyethyl)-RNA (MOE-RNA) nucleotides; and / or the amino acid sequence is derived from the wild-type sequence of a nucleic acid polymerase of the polB family; and / or 16. The nucleic acid polymerase of any one of claims 1, 13, and 15, wherein the amino acid sequence has at least 36% identity with the amino acid sequence of SEQ ID NO:

9.

19. 16. A method for making a non-DNA nucleotide polymer, comprising contacting a nucleic acid template with the nucleic acid polymerase of any one of claims 1, 13, and 15 under conditions that promote polymerization.

20. 20. The method of claim 19, wherein 2'OMe-RNA nucleotides and / or MOE-RNA nucleotides are provided during polymerization and the resulting non-DNA nucleotide polymer comprises said nucleotides.

21. 20. Use of the nucleic acid polymerase of any one of claims 1, 13 and 15 for the production of non-DNA nucleotide polymers.

22. 22. The use of claim 21, wherein the non-DNA nucleotide polymer comprises a 2'OMe-RNA nucleoside and / or a MOE-RNA nucleoside.

23. 16. A nucleic acid encoding the polymerase of any one of claims 1, 13, and 15.

24. 16. A host cell comprising the polymerase of any of claims 1, 13, and 15.

25. 24. A host cell comprising the nucleic acid of claim 23.

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