Modified nucleosides or nucleotides

• US 9,593,373 B2
• Filed: 03/15/2013
• Issued: 03/14/2017
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Liu, Xiaohai, Wu, Xiaolin, Smith, Geoffrey Paul
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/771,481
• Publication Number:
  US 20160002721A1
• Time in Patent Office: 1,460 Days
• Field of Search: 536/4.1, 536/23.1, 435/6.1, 435/91.1
• US Class Current: 1/1
• 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 purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′

-hydroxy protecting group forming a structure —

O—

C(R)₂N₃ covalently attached to the 3′

-carbon atom, wherein

R is independently selected from the group consisting of hydrogen, —

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 selected from hydrogen, or optionally substituted alkyl;

R² is halogen;

R³ is selected from hydrogen or optionally substituted alkyl;

each R⁴ and R⁵ is independently selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted aralkyl;

each R⁶ and R⁹ is selected from hydrogen, optionally substituted alkyl or halogen;

each R⁷, R⁸, R¹⁰ and R¹¹ is independently selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted aralkyl;

m is an integer of 0 to 3;

n is an integer of 0 to 3;

provided that the total of m+n equals to 3; and

p is an integer of 0 to 6;

provided that

at least one R is not hydrogen.

2. The modified nucleotide or nucleoside molecule of claim 1, wherein one of R is hydrogen and the other R is —

C(R¹)_m(R²)_n.

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

C(R¹)_m(R²)_n is selected from —

CHF₂, —

CH₂F, —

CHCl₂ or —

CH₂Cl.

4. The modified nucleotide or nucleoside molecule of claim 1, wherein one of R is hydrogen and the other R is —

C(═

O)OR³.

5. The modified nucleotide or nucleoside molecule of claim 1, wherein one of R is hydrogen and the other R is —

C(═

O)NR⁴R⁵, and wherein R⁴ is hydrogen and R⁵ is C_1-6 alkyl.

6. The modified nucleotide or nucleoside molecule of claim 1, wherein one of R is hydrogen and the other R is —

C(R⁶)₂O(CH₂)_pNR⁷R⁸.

7. The modified nucleotide or nucleoside molecule of claim 6, wherein both R⁶ are hydrogen.

8. The modified nucleotide or nucleoside molecule of claim 1, wherein one of R is hydrogen and the other R is —

C(R⁹)₂O-Ph-C(═

O)NR¹⁰R¹¹.

9. The modified nucleotide or nucleoside molecule of claim 8, wherein both R⁹ are hydrogen, and wherein R¹⁰ is hydrogen and R¹¹ is an amino substituted alkyl.

10. The modified nucleotide or nucleoside molecule of claim 1, wherein the 3′

-hydroxy protecting group is removed in a deprotecting reaction with a phosphine.

11. The modified nucleotide or nucleoside molecule of claim 10, wherein the phosphine is tris(hydroxymethyl)phosphine (THP).

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

13. 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.

14. The modified nucleotide or nucleoside molecule of claim 13, wherein the detectable label is a fluorophore.

15. 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 the growing complementary polynucleotide, wherein the incorporation of the modified nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide.

16. 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.

17. A method for 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 12;

and

detecting the identity of the modified nucleotide molecule.

18. The method of claim 17, wherein the identity of the modified nucleotide is determined by detecting the detectable label linked to the base.

19. The method of claim 17, wherein the 3′

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

20. A kit comprising a plurality of modified nucleotide or nucleoside molecules of claim 1, and packaging materials therefor.