MODIFIED NUCLEOSIDES OR NUCLEOTIDES

• US 20170166961A1
• Filed: 02/28/2017
• Published: 06/15/2017
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Smith, Geoffrey Paul, Wu, Xiaolin, Liu, Xiaohai
• Granted Patent: US 10,407,721 B2
• Time in Patent Office: Days
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Some embodiments described herein relate to modified nucleotide and nucleoside molecules with novel 3′-hydroxy protecting groups. Also provided herein are methods to prepare such modified nucleotide and nucleoside molecules and sequencing by synthesis processes using such modified nucleotide and nucleoside molecules.

Claims

1. A modified nucleotide or nucleoside molecule comprising a ribose or deoxyribose sugar moiety having a removable 3′

-hydroxy protecting group forming a structure —

O—

CH(R)N₃ covalently attached to the 3′

-carbon atom, wherein

R is selected from the group consisting of —

C(R¹)_m(R²)_n, —

C(═

O)OR³, —

C(═

O)NR⁴R⁵, —

C(R⁶)₂O(CH₂)_pNR⁷R⁸ and —

C(R⁹)₂O—

Ph—

C(═

O)NR¹⁰R¹¹;

R¹ is hydrogen, or optionally substituted alkyl;

R² is halogen;

R³ is hydrogen or optionally substituted alkyl;

each of R⁴ and R⁵ is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted aralkyl;

each of R⁶ and R⁹ is selected from the group consisting of hydrogen, optionally substituted alkyl and halogen;

each of R⁷, R⁸, R¹⁰ and R¹¹ is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted aralkyl;

m is an integer of 1 or 2;

n is an integer of 1 or 2;

provided that m+n is equal to 3; and

p is an integer of 0 to 6.

2. The modified nucleotide or nucleoside molecule of claim 1, wherein R is —

C(R¹)_m(R²)_n and wherein R¹ is hydrogen.

3. The modified nucleotide or nucleoside molecule of claim 2, wherein R is —

CHF₂, —

CH₂F, —

CHCl₂ or —

CH₂Cl.

4. The modified nucleotide or nucleoside molecule of claim 3, wherein R is —

CHF₂.

5. The modified nucleotide or nucleoside molecule of claim 3, wherein R is —

CH₂F.

6. The modified nucleotide or nucleoside molecule of claim 1, wherein R is —

C(═

O)NR⁴R⁵.

7. The modified nucleotide or nucleoside molecule of claim 1, wherein R⁴ is hydrogen and R⁵ is C_1-6 alkyl.

8. The modified nucleotide or nucleoside molecule of claim 1, wherein said modified nucleoside or nucleotide is linked to a detectable label via a cleavable linker or a non-cleavable linker.

9. The modified nucleotide or nucleoside molecule of claim 8, wherein the linker is cleavable.

10. The modified nucleotide or nucleoside molecule of claim 8, wherein the linker is acid labile, photo labile or comprises a disulfide linkage.

11. The modified nucleotide or nucleoside molecule of claim 8, wherein the detectable label is a fluorophore.

12. The modified nucleotide or nucleoside molecule of claim 1, wherein said 3′

-hydroxy protecting group is linked to a detectable label via a cleavable linker or a non-cleavable linker.

13. The modified nucleotide or nucleoside molecule of claim 12, wherein the linker is cleavable.

14. The modified nucleotide or nucleoside molecule of claim 12, wherein the linker is acid labile, photo labile or comprises a disulfide linkage.

15. The modified nucleotide or nucleoside molecule of claim 12, wherein the detectable label is a fluorophore.

16. A method of preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising incorporating a modified nucleotide molecule of claim 1 into a growing complementary polynucleotide, wherein the incorporation of the modified nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide.

17. The method of claim 15, wherein the incorporation of the modified nucleotide molecule is accomplished by a terminal transferase, a terminal polymerase or a reverse transcriptase.

18. A method of determining the sequence of a target single-stranded polynucleotide, comprising

monitoring the sequential incorporation of complementary nucleotides, wherein at least one complementary nucleotide incorporated is a modified nucleotide molecule of claim 8;

and

detecting the identity of the modified nucleotide molecule.

19. The method of claim 18, wherein the identity of the modified nucleotide is determined by detecting the detectable label.

20. The method of claim 18, wherein the 3′

-hydroxy protecting group and the detectable label are removed prior to introducing the next complementary nucleotide.

21. A kit comprising one or more modified nucleotide or nucleoside molecule of claim 1.