Modified nucleosides or nucleotides
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.
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) 2 N 3 covalently attached to the 3′-carbon atom, wherein
R is independently selected from the group consisting of hydrogen, —C(R 1 ) m (R 2 ) n , —C(═O)OR 3 , —C(═O)NR 4 R 5 , —C(R 6 ) 2 O(CH 2 ) p NR 7 R 8 and —C(R 9 ) 2 O-Ph-C(═O)NR 10 R 11 ;
R 1 is selected from hydrogen, or optionally substituted alkyl;
R 2 is halogen;
R 3 is selected from hydrogen or optionally substituted alkyl;
each R 4 and R 5 is independently selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted aralkyl;
each R 6 and R 9 is selected from hydrogen, optionally substituted alkyl or halogen;
each R 7 , R 8 , R 10 and R 11 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(R 1 ) m (R 2 ) n .
3. The modified nucleotide or nucleoside molecule of claim 2 , wherein —C(R 1 ) m (R 2 ) n is selected from —CHF 2 , —CH 2 F, —CHCl 2 or —CH 2 Cl.
4. The modified nucleotide or nucleoside molecule of claim 1 , wherein one of R is hydrogen and the other R is —C(═O)OR 3 .
5. The modified nucleotide or nucleoside molecule of claim 1 , wherein one of R is hydrogen and the other R is —C(═O)NR 4 R 5 , and wherein R 4 is hydrogen and R 5 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(R 6 ) 2 O(CH 2 ) p NR 7 R 8 .
7. The modified nucleotide or nucleoside molecule of claim 6 , wherein both R 6 are hydrogen.
8. The modified nucleotide or nucleoside molecule of claim 1 , wherein one of R is hydrogen and the other R is —C(R 9 ) 2 O-Ph-C(═O)NR 10 R 11 .
9. The modified nucleotide or nucleoside molecule of claim 8 , wherein both R 9 are hydrogen, and wherein R 10 is hydrogen and R 11 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.