WO2021205155A2 - Thymidines modifiées en c5 - Google Patents

Thymidines modifiées en c5 Download PDF

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Publication number
WO2021205155A2
WO2021205155A2 PCT/GB2021/050839 GB2021050839W WO2021205155A2 WO 2021205155 A2 WO2021205155 A2 WO 2021205155A2 GB 2021050839 W GB2021050839 W GB 2021050839W WO 2021205155 A2 WO2021205155 A2 WO 2021205155A2
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och
oligonucleotide
compound according
halo
group
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PCT/GB2021/050839
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WO2021205155A3 (fr
Inventor
Michael CHUN HAO CHEN
Michal ROBERT MATUSZEWSKI
Martin Fox
Gordon ROSS MCINROY
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Nuclera Nucleics Ltd
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Publication of WO2021205155A2 publication Critical patent/WO2021205155A2/fr
Publication of WO2021205155A3 publication Critical patent/WO2021205155A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the invention relates to 3'-aminooxy C5 modified thymidine nucleotides.
  • the invention also relates to a method of using said nucleotides in nucleic acid synthesis.
  • the invention further relates to a kit comprising the nucleotides, a terminal transferase enzyme and an initiator oligonucleotide.
  • Nucleic acid synthesis is vital to modern biotechnology. The rapid pace of development in the biotechnology arena has been made possible by the scientific community's ability to artificially synthesise DNA, RNA and proteins.
  • Nucleic acid synthesis is the process by which strands are assembled in the correct order of bases. Once assembled, the strands are frequently required to base-pair with their complementary strands. This involves the complementary pairing of nucleoside bases via hydrogen bonds, which is known as a base pair (bp).
  • the four nucleoside bases found in DNA are Adenine (A), Thymine (T), Cytosine (C), Guanine (G).
  • A Adenine
  • T Thymine
  • C Cytosine
  • G Guanine
  • U Uracil
  • A-T base pair is weaker than the C-G base pair (GC) due to having a lower number of hydrogen bonds (two vs three, respectively) and weaker stacking interactions.
  • GC C-G base pair
  • hybridisation between AT rich DNA strands is less favourable than GC rich strands - an effect that is clearly observable in the melting temperature (T m ).
  • T m melting temperature
  • DNA synthesis technology does not meet the demands of the biotechnology industry. Despite being a mature technology, it is practically impossible to synthesise a DNA strand greater than 200 nucleotides in length in viable yield, and most DNA synthesis companies only offer up to 120 nucleotides routinely.
  • an average protein-coding gene is of the order of 2000- 3000 contiguous nucleotides
  • a chromosome is at least a million contiguous nucleotides in length and an average eukaryotic genome numbers in the billions of nucleotides.
  • Known methods of DNA sequencing use template-dependent DNA polymerases to add 3'-reversibly terminated nucleotides to a growing double-stranded substrate.
  • each added nucleotide contains a dye, allowing the user to identify the exact sequence of the template strand.
  • this technology is able to produce strands of between 500-1000 bps long.
  • this technology is not suitable for de novo nucleic acid synthesis because of the requirement for an existing nucleic acid strand to act as a template.
  • TdT has been shown not to efficiently add nucleoside triphosphates containing 3'-0- reversibly terminating moieties for building up a nascent single-stranded DNA chain necessary for a de novo synthesis cycle.
  • a 3'-0- reversible terminating moiety would prevent a terminal transferase like TdT from catalysing the nucleotide transferase reaction between the 3'-end of a growing DNA strand and the 5'-triphosphate of an incoming nucleoside triphosphate.
  • the inventors have previously discovered certain modified nucleotides can be incorporated using terminal transferases. Modified nucleotides suitable for terminal transferase extension have been disclosed in for example PCT/GB2018/053305.
  • a common reversible terminator is the aminooxy (0-NH 2 ) group. The aminooxy group is converted to OH by treatment with nitrite.
  • nucleic acid synthesis In order to improve the quality of nucleic acid synthesis, it is desirable to shorten the length of nucleic acid fragments without reducing the melting temperature. Furthermore, reducing the dispiriting between the strength of AT and GC base pairs can improve downstream applications some as assembly of oligonucleotides into larger nucleic acid products. Whilst certain modifications to thymidine nucleosides are known to increase the melting temperature, these modifications have not previously been used in enzymatic synthesis.
  • the compounds described herein enable a method of increasing the AT base pair stability by modifying the 5-position pyrimidine group.
  • An aspect of the present invention relates to a compound according to Formula (la) or (lb): wherein, R 1 is a phosphate or polyphosphate group or salt thereof, optionally containing one or more sulfur atoms;
  • R 2 is H, halo, OH, NH 2 , COOH, COH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms;
  • R 3 is selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe.
  • a further aspect of the present invention relates to a compound according to Formula (lc) or (Id):
  • R 1 is a phosphate or polyphosphate group or salt thereof, optionally containing one or more sulfur atoms;
  • R 2 is H, halo, OH, NH 2 , COOH, COH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms;
  • R 3 is selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe; and R 4 is H or D.
  • a further aspect of the present invention also relates to a method of nucleic acid synthesis comprising reacting compound (la) or compound (lb) with an oligonucleotide sequence in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme.
  • TdT terminal deoxynucleotidyl transferase
  • a further aspect of the present invention also relates to a method of nucleic acid synthesis comprising reacting compound (lc) or compound (Id) with an oligonucleotide sequence in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme.
  • a further aspect of the present invention further relates to a kit comprising:
  • TdT terminal deoxynucleotidyl transferase
  • a further aspect of the present invention further relates to a kit comprising:
  • TdT terminal deoxynucleotidyl transferase
  • a further aspect of the present invention relates to an oligonucleotide according to Formula (2a) or (2b): wherein, R 1 is an oligonucleotide;
  • R 2 is H, halo, OH, NH 2 , COOH, COH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms;
  • R 3 is selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe.
  • a further aspect of the present invention relates to an oligonucleotide according to Formula (2c) or (2d): wherein, R 1 is an oligonucleotide;
  • R 2 is H, halo, OFI, N H 2 , COOFI, COH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms;
  • R 3 is selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe; and R 4 is H or D.
  • FIG. 1 Enzymatic incorporation of 2'-deoxy-3'-aminooxy-5-(3-hydroxy-l-butynyl)-uridine triphosphate (Tl) into a single stranded nucleic acid initiator.
  • An engineered terminal deoxynucleotidyl transferase (TdT) was used to incorporate either 2'-deoxythymidine triphosphate (T) or Tl into six nucleic acid sequences with different 3'-terminal sequence contexts (SEQ 1-6). Quantitative addition efficiency was observed in all cases.
  • Tl triphosphate is a suitable monomer for de novo enzymatic DNA synthesis.
  • This experiment also demonstrates that Tl behaves similarly to T in a de novo enzymatic DNA synthesis process.
  • a second addition was then performed, whereby the engineered TdT enzyme added either A, C, T, G, or Tl (all in 3'-aminooxy reversibly terminated form).
  • a cleavage solution containing uracil DNA glycosylase (UDG) and N,N'-dimethylethylene diamine (DMED) was then used to cleave DNA from the paramagnetic particles for analysis by denaturing polyacrylamide gel electrophoresis.
  • the DNA was visualised by virtue of the internal cyanine 3 dye. Lane 1: no addition control. Lanes 2 and 3: single addition of Tl triphosphate. Lanes 4-8: addition of Tl followed by A, C, T, G, or Tl for lanes 4 to 8 respectively.
  • the gel clearly shows quantitative addition of Tl to the solid support immobilised initiator strand. Furthermore, the gel demonstrates that multi-cycle de novo enzymatic DNA synthesis can be achieved when Tl forms part or the entirety of the synthesised sequence.
  • the A-T base pair is weaker than the C-G base pair due to having a lower number of hydrogen bonds (two vs three).
  • hybridisation between AT rich DNA strands is less favourable than GC rich strands - an effect that is clearly observable, for example through biophysical characterisation of the melting temperature (Tm).
  • Tm is defined as the temperature where 50% of oligonucleotide is duplexed with its perfect complement and 50% is free in solution. Tm is of critical importance for many biochemical techniques including PCR, in-situ hybridisation, and Southern blotting. In order for an AT rich region to attain an equal tm to a GC rich region, it must have a longer length.
  • An aspect of the present invention relates to a compound according to Formula (la) or (lb): wherein, R 1 is a phosphate or polyphosphate group or salt thereof, optionally containing one or more sulfur atoms;
  • R 2 is H, halo, OH, NH 2 , COOH, COH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms;
  • R 3 is selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe.
  • the oxime can be transformed into aminooxy as part of the unblocking process.
  • R 1 can be a phosphate or polyphosphate group.
  • the phosphate groups can be protonated or in salt form.
  • the phosphates can be entirely oxygen, or can contain one or more sulfur atoms.
  • R 1 can be a phosphate group.
  • R 1 can be a polyphosphate group.
  • R 1 can also be a phosphate or polyphosphate group selected from -(PO3) x (P02S) y (P0 3 ) z where x, y and z are independently 0-5 and and x+y+z is 1- 5.
  • R 1 can also be a phosphate or polyphosphate group having one or more sulfur atoms.
  • R 1 can be a phosphate group having one or more sulfur atoms.
  • R 1 can be a polyphosphate group having one or more sulfur atoms.
  • the sulfur atom can be in any position on any on the phosphate groups.
  • R 1 can further be a monophosphate, diphosphate, triphosphate, tetraphosphate, pentaphosphate, or (alpha-thio)triphosphate group.
  • R 1 can be a monophosphate group.
  • R 1 can be a diphosphate group.
  • R 1 can be a tetraphosphate group.
  • R 1 can be a pentaphosphate group.
  • R 1 can be an (alpha- thio)triphosphate group.
  • R 1 can be a triphosphate group.
  • a further embodiment of the present invention relates to a compound according to Formula (la) or (lb) wherein R 2 can be H, halo, OH, NH 2 , COOH, COH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms; and
  • R 2 can be H, F, OH, NH 2 , COOH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or F.
  • R 2 can be H, F, OH, NH 2 , Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or F.
  • R 2 can be H, F, OH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH or F.
  • R 2 can be H.
  • R 2 can be halo.
  • R 2 can be F.
  • R 2 can be Cl.
  • R 2 can be Br.
  • R 2 can be I.
  • R 2 can be NH 2 .
  • R 2 can be Ci_3 alkoxy.
  • R 2 can be OCH 3 .
  • R 2 can be OCH 2 CH 3 .
  • R 2 can be OCH 2 CH 2 CH 3 .
  • R 2 can be COOH.
  • R 2 can be COH.
  • R 2 can be Ci_ 3 alkyl optionally substituted with OH.
  • R 2 can be CH 2 OH.
  • R 2 can be CH 2 CH 2 OH.
  • R 2 can be CH 2 CH 2 CH 2 OH.
  • R 2 can be CH 2 CH(OH)CH 2 .
  • R 2 can be NH 2 .
  • R 2 can be OH or CH 2 OH.
  • R 2 can be OH.
  • R 2 can be CH 2 OH.
  • a further embodiment of the present invention relates to a compound according to Formula (la) or (lb) wherein R 3 can be selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe.
  • R 3 can be OH.
  • R 3 can be F.
  • R 3 can be OCH 3 .
  • R 3 can be OCH 2 CH 2 OMe.
  • R 3 can be H.
  • the compound of Formula (la) or (lb) of the present invention can be:
  • a further aspect of the present invention relates to a method of nucleic acid synthesis comprising reacting compound (la) or compound (lb) with an oligonucleotide sequence in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme.
  • the extended sequence can be treated with a nitrite salt.
  • the oxime can be converted to NH 2 prior to nitrite exposure, for example by hydrolysis of the oxime.
  • the terminal transferase or modified terminal transferase can be any enzyme capable of template independent strand extension.
  • the modified terminal deoxynucleotidyl transferase (TdT) enzyme can comprise amino acid modifications when compared to a wild type sequence or a truncated version thereof.
  • the terminal transferase can be the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in any species or the homologous amino acid sequence of RoIm, RoIb, RoIl, and RoIQ of any species or the homologous amino acid sequence of X family polymerases of any species.
  • Homologous refers to protein sequences between two or more proteins that possess a common evolutionary origin, including proteins from superfamilies in the same species of organism as well as homologous proteins from different species. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.
  • a variety of protein (and their encoding nucleic acid) sequence alignment tools may be used to determine sequence homology. For example, the Clustal Omega multiple sequence alignment program provided by the European Molecular Biology Laboratory (EMBL) can be used to determine sequence homology or homologous regions.
  • EMBL European Molecular Biology Laboratory
  • a further embodiment of the present invention relates to the oligonucleotide sequence comprising a solid-supported oligonucleotide sequence.
  • the oligonucleotide sequence comprises 2 or more nucleotides.
  • the oligonucleotide sequence can be between 10 and 500 nucleotides, such as between 20 and 200 nucleotides, in particular between 20 and 50 nucleotides long.
  • a further embodiment of the present invention relates to a method further comprising a reaction step with a nitrite salt.
  • the nitrate salt is sodium nitrite.
  • a further aspect of the present invention relates to a kit comprising:
  • TdT terminal deoxynucleotidyl transferase
  • a further aspect of the present invention relates to a compound according to Formula (lc) or (Id): wherein, R 1 is a phosphate or polyphosphate group or salt thereof, optionally containing one or more sulfur atoms;
  • R 2 is H, halo, OFI, N H 2 , COOFI, COH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms;
  • R 3 is selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe; and R 4 is H or D.
  • R 1 can be a phosphate or polyphosphate group.
  • the phosphate groups can be protonated or in salt form.
  • the phosphates can be entirely oxygen, or can contain one or more sulfur atoms.
  • R 1 can be a phosphate group.
  • R 1 can be a polyphosphate group.
  • R 1 can also be a phosphate or polyphosphate group selected from -(P03) x (P0 2 S) y (P03) z where x, y and z are independently 0-5 and and x+y+z is 1- 5.
  • R 1 can also be a phosphate or polyphosphate group having one or more sulfur atoms.
  • R 1 can be a phosphate group having one or more sulfur atoms.
  • R 1 can be a polyphosphate group having one or more sulfur atoms. The sulfur atom can be in any position on any on the phosphate groups.
  • R 1 can further be a monophosphate, diphosphate, triphosphate, tetraphosphate, pentaphosphate, or (alpha-thio)triphosphate group.
  • R 1 can be a monophosphate group.
  • R 1 can be a diphosphate group.
  • R 1 can be a tetraphosphate group.
  • R 1 can be a pentaphosphate group.
  • R 1 can be an (alpha- thio)triphosphate group.
  • R 1 can be a triphosphate group.
  • a further embodiment of the present invention relates to a compound according to Formula (lc) or (Id) wherein R 2 can be H, halo, OH, NH 2 , COOH, COH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms; and
  • R 2 can be H, F, OH, NH 2 , COOH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or F.
  • R 2 can be H, F, OH, NH 2 , Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or F.
  • R 2 can be H, F, OH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH or F.
  • R 2 can be H.
  • R 2 can be halo.
  • R 2 can be F.
  • R 2 can be Cl.
  • R 2 can be Br.
  • R 2 can be I.
  • R 2 can be NH 2 .
  • R 2 can be Ci_3 alkoxy.
  • R 2 can be OCH 3 .
  • R 2 can be OCH 2 CH 3 .
  • R 2 can be OCH 2 CH 2 CH 3 .
  • R 2 can be COOH.
  • R 2 can be COH.
  • R 2 can be Ci_ 3 alkyl optionally substituted with OH.
  • R 2 can be CH 2 OH.
  • R 2 can be CH 2 CH 2 OH.
  • R 2 can be CH 2 CH 2 CH 2 OH.
  • R 2 can be CH 2 CH(OH)CH 2 .
  • R 2 can be NH 2 .
  • R 2 can be OH or CH 2 OH.
  • R 2 can be OH.
  • R 2 can be CH 2 OH.
  • a further embodiment of the present invention relates to a compound according to Formula (lc) or (Id) wherein R 3 can be selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe.
  • R 3 can be OH.
  • R 3 can be F.
  • R 3 can be OCH 3 .
  • R 3 can be OCH 2 CH 2 OMe.
  • R 3 can be H.
  • a further embodiment of the present invention relates to a compound according to Formula (lc) or (Id) wherein R 4 can be H or D.
  • R 4 can be H.
  • R 4 can be D.
  • a further aspect of the present invention relates to an oligonucleotide according to Formula (2a) or (2b):
  • R 1 is an oligonucleotide
  • R 2 is H, halo, OH, NH 2 , Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms; and R 3 is selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe.
  • a further embodiment of the present invention relates to an oligonucleotide according to Formula (2a) or (2b) wherein R 2 can be H, halo, OH, NH 2 , Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms.
  • R 2 can be H.
  • R 2 can be halo.
  • R 2 can be F.
  • R 2 can be Cl.
  • R 2 can be Br.
  • R 2 can be I.
  • R 2 can be NH 2 .
  • R 2 can be Ci_ 3 alkoxy.
  • R 2 can be OCH 3 .
  • R 2 can be OCH 2 CH 3 .
  • R 2 can be OCH 2 CH 2 CH 3 .
  • R 2 can be Ci_ 3 alkyl optionally substituted with OH.
  • R 2 can be CH 2 OH.
  • R 2 can be CH 2 CH 2 OH.
  • R 2 can be CH 2 CH 2 CH 2 OH.
  • R 2 can be CH 2 CH(OH)CH 2 .
  • R 2 can be NH 2 .
  • R 2 can be OH or CH 2 OH.
  • R 2 can be OH.
  • R 2 can be CH 2 OH.
  • a further embodiment of the present invention relates to an oligonucleotide according to Formula (2a) or (2b) wherein R 3 can be selected from H, OH, F, or OCH 3 .
  • R 3 can be OH.
  • R 3 can be F.
  • R 3 can be OCH 3 .
  • R 3 can be H.
  • a further aspect of the present invention relates to an oligonucleotide according to Formula (2c) or (2d): wherein, R 1 is an oligonucleotide;
  • R 2 is H, halo, OH, NH 2 , COOH, COH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms;
  • R 3 is selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe; and R 4 is H or D.
  • a further embodiment of the present invention relates to an oligonucleotide according to Formula (2c) or (2d) wherein R 2 can be H, halo, OH, NH 2 , Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms.
  • R 2 can be H.
  • R 2 can be halo.
  • R 2 can be F.
  • R 2 can be Cl.
  • R 2 can be Br.
  • R 2 can be I.
  • R 2 can be NH 2 .
  • R 2 can be Ci_ 3 alkoxy.
  • R 2 can be OCH 3 .
  • R 2 can be OCH 2 CH 3 .
  • R 2 can be OCH 2 CH 2 CH 3 .
  • R 2 can be Ci_ 3 alkyl optionally substituted with OH.
  • R 2 can be CH 2 OH.
  • R 2 can be CH 2 CH 2 OH.
  • R 2 can be CH 2 CH 2 CH 2 OH.
  • R 2 can be CH 2 CH(OH)CH 2 .
  • R 2 can be NH 2 .
  • R 2 can be OH or CH 2 OH.
  • R 2 can be OH.
  • R 2 can be CH 2 OH.
  • a further embodiment of the present invention relates to an oligonucleotide according to Formula (2c) or (2d) wherein R 3 can be selected from H, OH, F, or OCH 3 .
  • R 3 can be OH.
  • R 3 can be F.
  • R 3 can be OCH 3 .
  • R 3 can be H.
  • a further embodiment of the present invention relates to a compound according to Formula (2c) or (2d) wherein R 4 can be H or D.
  • R 4 can be H.
  • R 4 can be D.
  • Described herein is a process of nucleic acid synthesis using the compounds described herein.
  • the process uses a nucleic acid polymerase, which may be a template independent polymerase or a template dependent polymerase to add a single nucleotide to one or more nucleic acid strands.
  • the strands may be immobilised on a solid support.
  • the process involves cleaving the 3'-aminooxy group and adding a further nucleotide, the base of which may or may not be T.
  • nucleic acid synthesis comprising:
  • extension reagents comprising a polymerase or terminal deoxynucleotidyl transferase (TdT) and a compounds according to Formula (la) or (lb):
  • R 1 is a phosphate or polyphosphate group or a salt thereof, optionally containing one or more sulfur atoms;
  • R 2 is H, halo, OH, NH 2 , COOH, COH, Ci_ 3 alkoxy, Ci_ 3 alkyl optionally substituted with OH, NH 2 or halo atoms;
  • R 3 is selected from H, OH, F, OCH 3 or OCH 2 CH 2 OMe.
  • the nucleic acids synthesised can be any sequence. One or more, possibly all, of the thymine bases will have a modification at the 5-position. A population of different sequences can be synthesised in parallel.
  • heterocyclic bases have exocyclic NH 2 groups, for example cytidine, adenine or guanine
  • these groups can optionally be masked by an orthogonal masking agent.
  • the amine masked nitrogenous heterocycles may be N4-masked cytidine, N6-amine masked adenine and N2-amine masked guanine.
  • the masking may be for example an azido (N 3 ) group.
  • references herein to an "amine masking group” refer to any chemical group which is capable of generating or “unmasking" an amine group which is involved in hydrogen bond base-pairing with a complementary base. Most typically the unmasking will follow a chemical reaction, most suitably a simple, single step chemical reaction.
  • the amine masking group will generally be orthogonal to the 3'-0-NH 2 blocking group in order to allow selective removal.
  • the purine compounds may be selected from: where R 1 and R 3 are as defined herein.
  • This embodiment has the advantage of reversibly masking the -NH 2 group. While blocked in the -N 3 state, the base (B) is impervious to deamination (e.g., deamination in the presence of sodium nitrite). The base (B) in the N-blocked form is incapable of forming secondary structures via base pairing. Thus even blocking a subset of the free amino groups in the nucleic acid polymer improves the availability of the 3'-end for further extension.
  • the canonical cytosine, adenine, guanine can be respectively recovered from 4-azido cytosine, 6-azido adenine and 2-azido guanine by exposure to a reducing agent (e.g., TCEP).
  • a reducing agent e.g., TCEP
  • nucleic acid synthesis may be readily applied to methods of enzymatic nucleic acid synthesis which are well known to the person skilled in the art.
  • Non-limiting methods of nucleic acid synthesis may be found in WO 2016/128731, WO 2016/139477, WO 2017/009663, GB 1613185.6 and GB 1714827.1, the contents of each of which are herein incorporated by reference.
  • Enzymatic nucleic acid synthesis is defined as any process in which a nucleotide is added to a nucleic acid strand through enzymatic catalysis in the presence or absence of a template.
  • a method of enzymatic nucleic acid synthesis could include non-templated de novo nucleic acid synthesis utilizing a PoIX family polymerase, such as terminal deoxynucleotidyl transferase, and reversibly terminated 2'-deoxynucleoside 5'-triphosphates or ribonucleoside 5'-triphosphate.
  • Another method of enzymatic nucleic acid synthesis could include templated nucleic acid synthesis, including sequencing-by-synthesis.
  • Reversibly terminated enzymatic nucleic acid synthesis is defined as any process in which a reversibly terminated nucleotide is added to a nucleic acid strand through enzymatic catalysis in the presence or absence of a template.
  • the method of enzymatic nucleic acid synthesis is selected from a method of reversibly terminated enzymatic nucleic acid synthesis and a method of templated and non-templated de novo enzymatic nucleic acid synthesis.
  • nucleoside triphosphates refer to a molecule containing a nucleoside (i.e. a base attached to a deoxyribose or ribose sugar molecule) bound to three phosphate groups.
  • nucleoside triphosphates that contain deoxyribose are: deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) or deoxythymidine triphosphate (dTTP).
  • nucleoside triphosphates examples include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) or uridine triphosphate (UTP).
  • ATP adenosine triphosphate
  • GTP guanosine triphosphate
  • CTP cytidine triphosphate
  • UDP uridine triphosphate
  • Other types of nucleosides may be bound to three phosphates to form nucleoside triphosphates, such as naturally occurring modified nucleosides and artificial nucleosides.
  • references herein to '3'-blocked nucleoside triphosphates' refer to nucleoside triphosphates (e.g., dATP, dGTP, dCTP or dTTP) which have an additional group on the 3' end which prevents further addition of nucleotides, i.e., by replacing the 3'-OH group with a protecting group.
  • the protecting group is NH 2 or a protected version thereof.
  • references herein to a 'DNA initiator sequence' refer to a small sequence of DNA which the 3'- blocked nucleoside triphosphate can be attached to, i.e., DNA will be synthesised from the end of the DNA initiator sequence.
  • the initiator sequence is between 5 and 10 nucleotides long, such as between 10 and 60 nucleotides long, in particular between 20 and 50 nucleotides long.
  • the initiator sequence is single-stranded. In an alternative embodiment, the initiator sequence is double-stranded. In another embodiment, the initiator sequence has a single stranded portion and a double stranded portion. It will be understood by persons skilled in the art that a 3'-overhang (i.e., a free 3'-end) allows for efficient addition.
  • the initiator sequence is immobilised on a solid support. This allows the enzyme and the cleaving agent to be removed without washing away the synthesised nucleic acid.
  • the initiator sequence may be attached to a solid support stable under aqueous conditions so that the method can be easily performed via a flow setup.
  • the initiator sequence is immobilised on a solid support via a reversible interacting moiety, such as a chemically-cleavable linker, an antibody/immunogenic epitope, a biotin/biotin binding protein (such as avidin or streptavidin), or glutathione-GST tag. Therefore, in a further embodiment, the method additionally comprises extracting the resultant nucleic acid by removing the reversible interacting moiety in the initiator sequence, such as by incubating with proteinase K.
  • a reversible interacting moiety such as a chemically-cleavable linker, an antibody/immunogenic epitope, a biotin/biotin binding protein (such as avidin or streptavidin), or glutathione-GST tag. Therefore, in a further embodiment, the method additionally comprises extracting the resultant nucleic acid by removing the reversible interacting moiety in the initiator sequence, such as by incubating with proteinase K
  • the initiator sequence contains a base or base sequence recognisable by an enzyme.
  • a base recognised by an enzyme such as a glycosylase, may be removed to generate an abasic site which may be cleaved by chemical or enzymatic means.
  • An example of such a glycosylase system includes the presence of a uracil base in the initiator sequence, which may be excised with uracil DNA glycosylase (UDG) to leave an abasic site which may be cleaved with, for example, basic solutions, organic amines, or an endonuclease (such as endonuclease VIII), to release a nucleic acid bearing a 5'-phosphate into solution.
  • a base sequence may be recognised and cleaved by a restriction enzyme.
  • the initiator sequence is immobilised on a solid support via an orthogonal chemically-cleavable linker, such as a disulfide, allyl, or azide-masked hemiaminal ether linker. Therefore, in one embodiment, where the N-masking group is not azido, the method additionally comprises extracting the resultant nucleic acid by cleaving the chemical linker through the addition of tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) for a disulfide linker; palladium complexes or an allyl linker; or TCEP for an azide-masked hemiaminal ether linker.
  • TCEP tris(2-carboxyethyl)phosphine
  • DTT dithiothreitol
  • the resultant nucleic acid is extracted and amplified by polymerase chain reaction using the nucleic acid bound to the solid support as a template.
  • the initiator sequence could therefore contain an appropriate forward primer sequence and an appropriate reverse primer could be synthesised.
  • the terminal deoxynucleotidyl transferase (TdT) of the invention is added in the presence of an extension solution comprising one or more buffers (e.g., Tris or cacodylate), one or more salts (e.g., Na + , K + , Mg 2+ , Mn 2+ , Cu 2+ , Zn 2+ , Co 2+ , etc. all with appropriate counterions, such as Cl) and inorganic pyrophosphatase (e.g., the Saccharomyces cerevisiae homolog).
  • buffers e.g., Tris or cacodylate
  • salts e.g., Na + , K + , Mg 2+ , Mn 2+ , Cu 2+ , Zn 2+ , Co 2+ , etc. all with appropriate counterions, such as Cl
  • inorganic pyrophosphatase e.g., the Saccharomyces cerevisiae homolog
  • an inorganic pyrophosphatase helps to reduce the build-up of pyrophosphate due to nucleoside triphosphate hydrolysis by TdT. Therefore, the use of an inorganic pyrophosphatase has the advantage of reducing the rate of (1) backwards reaction and (2) TdT strand dismutation.
  • step (b) is performed at a pH range between 5 and 10. Therefore, it will be understood that any buffer with a buffering range of pH 5-10 could be used, for example cacodylate, Tris, HEPES orTricine, in particular cacodylate orTris.
  • the compounds of the invention can be used on a device for nucleic acid synthesis.
  • a solid support in the form of, for example, a planar array and further a plurality of beads onto which a plurality of immobilized initiation oligonucleotide sequences are attached.
  • the beads may be porous and a portion of the, optionally porous, beads are selected as anchors and unselected beads are exposed to harvest solution to cleave them from their solid support to release the oligonucleotide sequences into solution.
  • the term solid support can refer to an array having a plurality of beads which may or may not be immobilised.
  • the oligonucleotides may be attached to, or removed from beads whilst on the array.
  • the immobilised oligonucleotide may be attached to a bead, which remains in a fixed position on the array whilst other beads in other locations are subject to cleavage conditions to detach the oligonucleotides from the beads (the beads may or may not be immobilised).
  • the solid support can take the form of a digital microfluidic device.
  • Digital microfluidic devices consist of a plurality of electrodes arranged on a surface.
  • a dielectric layer e.g., aluminum oxide
  • a hydrophobic coating e.g., perfluorinated hydrocarbon polymer
  • the electrodes may be hardwired or formed from an active matrix thin film transistor (AM-TFT).
  • AM-TFT active matrix thin film transistor
  • the solid support can take the form of a digital microfluidic device.
  • Digital microfluidic devices consist of a plurality of electrodes arranged on a surface. These electrodes can be addressed in a passive manner or by active matrix methods. Passive addressing is a direct address where actuation signals are directly applied on individual electrode (for example by means of a hard-wired connection to that electrode in a single layer or multilayer fashion such as a printed circuit board, PCB).
  • PCB printed circuit board
  • direct drive methods is the inability to process large numbers of droplets due to difficulties in addressing large numbers of direct drive electrodes.
  • MxN electrodes can be controlled by M+N pins, significantly reducing the number of control pins.
  • An AM- TFT digital microfluidic device comprises a dielectric layer (e.g., aluminum oxide) deposited over the electrode layer on the thin-film transistor layer followed by a hydrophobic coating (e.g., perfluorinated hydrocarbon polymer) atop the dielectric layer.
  • a dielectric layer e.g., aluminum oxide
  • a hydrophobic coating e.g., perfluorinated hydrocarbon polymer
  • aqueous droplets may be actuated across the surface immersed in oil, air, or another fluid.
  • Enzymatic oligonucleotide synthesis can be deployed on a digital microfluidic device in several ways.
  • An initiator oligonucleotide can be immobilized via the 5'-end on super paramagnetic beads or directly to the hydrophobic surface of the digital microfluidic device.
  • a plurality of distinct positions containing immobilized initiator oligonucleotides on the digital microfluidic device may be present (henceforth named synthesis zones).
  • Solutions required for enzymatic oligonucleotide synthesis are then dispensed from multiple reservoirs onto the device.
  • an addition solution containing the components necessary for the TdT-mediated incorporation of reversibly terminated nucleoside 5'-triphosphates onto immobilized initiator oligonucleotides can be dispensed from a reservoir in droplets and actuated to the aforementioned positions containing immobilized initiator oligonucleotides.
  • each reservoir (and thus each droplet containing addition solution) can contain a distinct nitrogenous base reversibly terminated nucleoside 5'-triphosphate identity or a mixture thereof in order to control the sequence synthesized on aforementioned positions containing immobilized initiator oligonucleotides.
  • the method can be implemented on continuous flow microfluidic devices.
  • One such device consists of a surface with a plurality of microwells each containing a bead. On said bead, an oligonucleotide initiator can be immobilized.
  • each microwell can contain an electrode to perform electrochemistry.
  • the use of the modified thymine bases improves the quality of the synthesised strands due to lowering the length required in order to obtain a hybridising strand.
  • T1 -oxime Scheme 3 Oxime deprotection of Tl-oxime to yield Tl. Prior to use, the oxime can be removed by incubation of the triphosphate Tl-oxime in a solution of 1 M sodium acetate pH 5.5, 1.5% w/v methoxylamine, and ultrapure water for 60 minutes at room temperature.
  • A3 ⁇ 4Q ZnCI 2 Zinc chloride (0.49 g, 3.6 mmol) was placed in a 50 mL round bottomed flask. Acetic anhydride (8.0 mL, 86 mmol) was added, then 3-butyn-l-ol (5.4 mL, 5.0 g, 71 mmol) was added to the suspension over 20 minutes. The solution was stirred at room temperature for 30 minutes, then water (30 mL) was added over 5 minutes. The mixture was stirred vigorously for 30 minutes, when the layers were separated. The organic phase was washed with water (30 mL) and sodium bicarbonate solution (10 mL).
  • the solvent was evaporated from the bulk using a rotary evaporator, then 1 M citric acid (10 mL) and. ethyl acetate (30 mL) were added, the mixture was shaken and the layers were separated. The organic layer was washed with 1 M citric acid, (2 x 10 mL) and saturated sodium bicarbonate solution (10 mL). The organic layer was dried (Na 2 S0 4 ) and filtered. The solvent was removed using a rotary evaporator to give a white solid (1.31 g), which was dissolved in hot acetone (15 mL). The solution was allowed to cool to room temperature, then heptane (15 mL) was added over 90 minutes.
  • the white suspension was stirred at room temperature for 1 h, filtered and the solid was washed with heptane-acetone (1:1, 2 x 5 mL) and dried under vacuum, to give 5 '(O)-tert- butyldimethylsilyl-2'-deoxy-5-iodouridine as a fine white solid (652 mg). Additional heptane (20 mL) was added to the filtrate; this was stirred at room temperature for 30 minutes, the filtered and the solid was washed with heptane-acetone (3:1, 2 x 5 mL) and dried under vacuum, yield of second crop of as a fine white solid (257 mg). Additional solid precipitated from the filtrate.
  • the pale yellow solution was stirred for a further 1 h, when the solvent was removed using a rotary evaporator. Toluene (10 mL) was added and the solvent was evaporated again. Toluene (20 mL) was added, and the solution was washed with saturated sodium bicarbonate solution (10 mL). The mixture was shaken and the layers were separated. The organic layer was washed with saturated sodium bicarbonate solution (10 mL), dried (Na 2 S0 4 ), filtered and the solution was concentrated using a rotary evaporator to ⁇ 5 mL. The pale yellow solution was placed in the freezer (-20°C) for 16 h, then allowed to warm to room temperature, and the solvent was evaporated again.
  • the suspension was stirred at 0-5°C for 30 minutes, then allowed to warm to room temperature. After lh, the reaction was quenched with water (0.1 mL), and the solution was stirred for a further 30 minutes. Toluene (15 mL) was added, followed by saturated sodium bicarbonate solution (15 mL), the mixture was shaken, the layers were separated and the organic phase was washed with saturated sodium bicarbonate solution (10 mL + 2 x 5 mL), dried (Na 2 S0 4 ) and filtered. The solution was concentrated using a rotary evaporator to about 10 mL, then seeded with a small quantity of triphenylphosphine oxide-diisopropylhydrazine dicarboxylate complex.
  • iro/is-Dichlorobis(triphenylphosphine)palladium(ll) 7 mg, 0.0096 mmol
  • copper(l) iodide 7 mg, 0.053 mmol
  • the reaction flask was purged with nitrogen and heated in a heating block at 60°C for 1 h. After cooling, most of the solvent was removed using a rotary evaporator, then ethyl acetate (5 mL) and 5% disodium EDTA solution (5 mL) were added. The layers were separated, and the organic phase was washed with disodium EDTA (5 mL).
  • the organic phase was dried (Na 2 S0 4 ), filtered and the solvent was removed using a rotary evaporator.
  • the product was purified on a short silica column eluting with heptane-ethyl acetate (1:1).
  • the solid product was dissolved in toluene (2 mL). Heptane (3 mL) was added while stirring over 15 minutes.
  • Ethoxytrimethylsilane (1.0 mL, 6.5 mmol) was added over 2 minutes, the solution was stirred for 30 minutes then saturated sodium bicarbonate solution (5 mL) and ethyl acetate (5 mL) were added. The phases were separated. The aqueous phase was extracted with ethyl acetate (2 x 5 mL), the combined organic phases were dried (Na 2 S0 4 ) and filtered and the solvent was removed using a rotary evaporator to give a white solid.
  • the suspension was stirred at room temperature for 1 h, when most of the solvent was removed using a rotary evaporator (bath temperature 30°C).
  • the residue was dissolved in water (2 mL) and split between two 50 mL centrifuge tubes.
  • a white emulsion formed.
  • the upper layer was decanted to leave the lower, immiscible oily phase. This was dissolved in water (2 x 0.5 mL).
  • the oily residues were dissolved in water (total volume 2 mL) and the crude triphosphate solution obtained was purified by reverse phase HPLC using a Supelco Ascentis C18 column (25 cm x 10 mm, 5 pm), flow rate 3 mL/min, and a gradient of A: 100 mM triethylammonium bicarbonate pH 7.5, B: Acetonitrile; A to 30%B over 32 minutes (8 runs).
  • the product-containing fractions were combined and the solvent was evaporated, then methanol (10 mL) was added to the residue and the solvent was evaporated using a rotary evaporator.
  • Tl 2'-deoxy-3'-aminooxy-5-(3-hydroxy-l-butynyl)-uridine triphosphate
  • TdT An engineered terminal deoxynucleotidyl transferase (TdT) was used to incorporate Tl into six nucleic acid sequences (SEQs 1 to 6) with different 3'-terminal sequence contexts (see Figure 1). Quantitative addition efficiency was observed in all cases. This demonstrates that the Tl triphosphate is a suitable monomer for de novo enzymatic DNA synthesis.
  • Tl 2'-deoxy-3'-aminooxy-5-(3-hydroxy-l- butynylj-uridine triphosphate
  • Initiator oligonucleotide of SEQ 7 was immobilised on super paramagnetic particles to facilitate retention through multiple cycles of DNA synthesis.
  • An engineered TdT enzyme mediated the first addition of Tl to the initiator oligonucleotide.
  • Sodium nitrite in acetate buffer was then employed to deblock the 3'-aminooxy reversible terminator, revealing a 3'-hydroxyl competent for further addition.
  • a second addition was then performed, whereby the engineered TdT enzyme added either A, C, T, G, or Tl (all in 3'-aminooxy reversibly terminated form).
  • a cleavage solution containing uracil DNA glycosylase (UDG) and N,N'- dimethylethylene diamine (DMED) was then used to cleave DNA from the paramagnetic particles for analysis by denaturing polyacrylamide gel electrophoresis.
  • the DNA was visualised by virtue of the internal cyanine 3 dye (see figure 2). Lane 1: no addition control. Lanes 2 and 3: single addition of Tl triphosphate. Lanes 4-8: addition of Tl followed by A, C, T, G, or Tl for lanes 4 to 8 respectively.
  • an asterisk (*) indicates a phosphorothioate linkage and /icy3/ indicates an internal cyanine 3 dye.

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Abstract

La présente invention concerne un composé selon la formule (1c) ou (1d) : dans laquelle, R1, R2, R3 et R4 sont tels que décrits dans la description.
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WO2016128731A1 (fr) 2015-02-10 2016-08-18 Nuclera Nucleics Ltd Nouvelle utilisation
WO2016139477A1 (fr) 2015-03-03 2016-09-09 Nuclera Nucleics Ltd Procédé de synthèse d'acide nucléique par l'intermédiaire d'un 3'-o-azidométhylnucléotide triphosphate
WO2017009663A1 (fr) 2015-07-15 2017-01-19 Nuclera Nucleics Ltd Procédé de déprotection de l'éther azidométhylique

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US8034923B1 (en) * 2009-03-27 2011-10-11 Steven Albert Benner Reagents for reversibly terminating primer extension
US11085076B2 (en) * 2015-09-28 2021-08-10 The Trustees Of Columbia University In The City Of New York Synthesis of novel disulfide linker based nucleotides as reversible terminators for DNA sequencing by synthesis
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WO2016128731A1 (fr) 2015-02-10 2016-08-18 Nuclera Nucleics Ltd Nouvelle utilisation
WO2016139477A1 (fr) 2015-03-03 2016-09-09 Nuclera Nucleics Ltd Procédé de synthèse d'acide nucléique par l'intermédiaire d'un 3'-o-azidométhylnucléotide triphosphate
WO2017009663A1 (fr) 2015-07-15 2017-01-19 Nuclera Nucleics Ltd Procédé de déprotection de l'éther azidométhylique

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