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US-12624374-B1 - Processes using nucleoside triphosphates with stable aminoxy groups

US12624374B1US 12624374 B1US12624374 B1US 12624374B1US-12624374-B1

Abstract

This invention claims processes that append a single nucleotide having a 3′-ONH 2 moiety to the 3′-ends of an oligonucleotide primer using 3′-deoxynucleoside triphosphates that have, instead of a 3′-OH moiety, a 3′-ONH 2 moiety, where the nucleotides contain both standard and non-standard nucleobases, and where as a key claim limitation, substantially no hydroxylamine is present in the solutions used in the claimed processes.

Inventors

  • Nicole A. Leal
  • Steven A Benner

Assignees

  • FIREBIRD BIOMOLECULAR SCIENCES

Dates

Publication Date
20260512
Application Date
20210913

Claims (17)

  1. 1 . A process that adds to an oligonucleotide primer a single nucleotide that has a 3′-ONH 2 moiety instead of a 3′-OH moiety, wherein said process comprises contacting an oligonucleotide primer in an aqueous buffered solution with an enzyme having terminal transferase activity and a compound having the structure: or one of its ionized forms, wherein Z is either H, OH, or OCH 3 , B is a heterocycle selected from the group consisting of wherein Su indicates the point of attachment of the heterocycle to the sugar, R is either H, CH 3 , or a functionalized side chain, and X is N, and wherein the concentration of hydroxylamine in said aqueous buffered solution that is less than 1 micromolar.
  2. 2 . The process of claim 1 , wherein said solution also contains additional dissolved alkoxylamine.
  3. 3 . The process of claim 2 , wherein said heterocycle B is selected from the group consisting of thymine, uracil, cytosine, guanine, adenine, and isocytosine.
  4. 4 . The process of claim 2 , wherein said heterocycle B is selected from the group consisting of thymine and isocytosine.
  5. 5 . The process of claim 2 , wherein appended to heterocycle B is a linker that comprises either a disulfide moiety or a 1,2-diol moiety.
  6. 6 . The process of claim 2 , wherein said heterocycle is and wherein Su indicates the point of attachment of the heterocycle to the sugar.
  7. 7 . The process of claim 2 , wherein said heterocycle is wherein Su indicates the point of attachment of the heterocycle to the sugar, and R is either H, CH 3 , or a functionalized side chain.
  8. 8 . The process of claim 2 , wherein the concentration of the dissolved alkoxylamine is between 10 and 50 micromolar, and is delivered in a sequence where the enzyme is first contacted with the primer, then contacted with the additional alkoxylamine, and then contacted with the triphosphates.
  9. 9 . The process of claim 2 , wherein said enzyme is terminal deoxynucleotide transferase.
  10. 10 . The process of claim 2 , wherein said enzyme is a DNA polymerase, an RNA polymerase, a polyA polymerase, polyU polymerase, or a terminal transferase, and said oligonucleotide is hybridized to a template oligonucleotide.
  11. 11 . The process of claim 1 , wherein said heterocycle B is selected from the group consisting of thymine, uracil, cytosine, guanine, adenine, and isocytosine.
  12. 12 . The process of claim 1 , wherein said heterocycle B is selected from the group consisting of thymine and isocytosine.
  13. 13 . The process of claim 1 , wherein appended to heterocycle B is a linker that comprises either a disulfide moiety or a 1,2-diol moiety.
  14. 14 . The process of claim 1 , wherein said heterocycle is and wherein Su indicates the point of attachment of the heterocycle to the sugar.
  15. 15 . The process of claim 1 , wherein said heterocycle is wherein Su indicates the point of attachment of the heterocycle to the sugar, and R is either H, CH 3 , or a functionalized side chain.
  16. 16 . The process of claim 1 , wherein said enzyme is terminal deoxynucleotide transferase.
  17. 17 . The process of claim 1 , wherein said enzyme is a DNA polymerase, an RNA polymerase, a polyA polymerase, polyU polymerase, or a terminal transferase, and said oligonucleotide is hybridized to a template oligonucleotide.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This is a continuation-in-part of U.S. patent application Ser. No. 16/679,501, filed 11 Nov. 2019, currently pending, which was a continuation in part of U.S. patent application Ser. No. 15/460,475, filed 16 Mar. 2017 now U.S. patent Ser. No. 10/472,383, and U.S. patent application Ser. No. 15/786,086, filed 17 Oct. 2017 now U.S. patent Ser. No. 10/654,841. U.S. patent application Ser. No. 15/786,086 is a continuation in part of U.S. patent application Ser. No. 15/475,694, filed 31 Mar. 2017, and now abandoned. This is also a continuation-in-part of U.S. patent application Ser. No. 16/887,951, filed 29 May 2020, pending. This is also a continuation-in-part of U.S. patent application Ser. No. 16/679,887, filed 29 May 2020, pending. STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED RESEARCH This invention was made with government support under R41GM119494 awarded by the National Institutes of General Medical Sciences. The government has certain rights in the invention. THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT Not applicable INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC None BACKGROUND OF THE INVENTION (1) Field of the Invention This invention relates to the field of nucleic acid chemistry, and more specifically to DNA and DNA-like molecules that have a 3′-ONH2 group rather than the 3′-OH group that is found in standard DNA, DNA with non-standard nucleobases, and DNA-like molecules (collectively hereinafter “DNA”). Still more specifically, this invention relates to enzymatic processes that attach, to the end of a standard DNA, RNA, and/or DNA-like or RNA-like molecule, a nucleotide that has its 3′-OH group substantially completely replaced by a 3′-ONH2 group, using a triphosphate analog in an aqueous solution that lacks hydroxylamine. This invention also relates to processes where a DNA polymerase, a reverse transcriptase, a polynucleotide polymerase (e.g. polyA polymerase or polyU polymerase [Church, G. M., Wiegand, D. J., Kohnan, R. E., Kuru, E., Ricttichier, J., Conway, N. (2020) Enzymatic RNA Synthesis, WO/2020/077227 A2][Heinisch, T., Champion, E., Sune, E., Soskine, M. (2021) Template Free Enzymatic synthesis of polynucleotides using Poly(A) and Poly (U) polymerases, WO/2021/018919], or a terminal deoxynucleotide transferase uses a nucleoside triphosphate having a 3′-ONH2 group to add a 3′-aminoxy-2′,3′-dideoxynucleotide to the 3′-end of a DNA or DNA-like molecule. (2) Description of Related Art Well-known in the art are useful processes that require that the enzymatic extension of a DNA, DNA-like, or RNA oligonucleotide (hereinafter a primer) be terminated after introduction of just a single nucleotide at the 3′-end. This extension may be templated, as in the primer-extension processes that are catalyzed by DNA polymerases, RNA polymerases, or reverse transcriptases. Here, successful termination after the addition of just one nucleotide underlies many DNA sequencing architectures, especially those known to use “cyclic reversible termination”. Termination of template-guided extension after the addition of a single nucleotide is frequently achieved by contacting the enzyme to analog of a nucleoside triphosphate where the nucleoside has been altered so as to no longer have a free 3′-hydroxyl group. Also well known in the art are processes where extension in not templated. Here, a common enzyme to catalyze the process is a terminal deoxynucleotide transferase (TdT). Termination after addition of just a single nucleotide is used in many DNA synthesizing architectures. Well known among these analogs are triphosphates where the 3′-hydroxyl group is replaced by a hydrogen atom to generate 2′,3′-dideoxynucleoside triphosphates. These are substrates for many polymerases, including many modified polymerases. In forms that carry side chains carrying reporter groups, these have long been used in DNA sequencing processes. Since no convenient method is available to replace the 3′-H by a 3′-OH group on an oligonucleotide, the termination of the oligonucleotide extension process in the presence of a 2′,3′-dideoxynucleoside triphosphate analog is said to be irreversible. Other nucleoside and oligonucleotide derivatives lacking the standard 3′-OH have functionality that can later be converted to a 3′-OH group under conditions that do not damage oligonucleotides. This allows template-directed primer extension to be terminated “reversibly”. For example, various patents, including U.S. Pat. Nos. 7,544,794, 8,034,923, and 8,212,020, disclosed that a 3′-O—NH2 group may be used a reversibly terminating moiety. These are referred to as 3′-aminoxy-2′,3′-dideoxynucleosides, -tides, and triphosphates. After a nucleotide having a 3′-O—NH2 group is added to the 3′-end of an oligonucleotide primer, further polymerase-catalyzed extension cannot occur. This terminating 3′-O—NH2 group may not be removed, allowing its reactivity to be used for a vari