METHODS AND COMPOSITIONS FOR CONTINUOUS SINGLE-MOLECULE NUCLEIC ACID SEQUENCING BY SYNTHESIS WITH FLUOROGENIC NUCLEOTIDES

1 . A method for sequencing a nucleic acid, said method comprising the steps of:

a) disposing in an optionally sealed microreactor a mixture in solution phase comprising a single copy of a target nucleic acid, a nucleic acid replicating catalyst, and a mixture of nucleotides, wherein said mixture of nucleotides comprises a first nucleotide comprising a first label that is substantially non-fluorescent until after incorporation of said first nucleotide into a nucleic acid based on complementarity to said target nucleic acid;

b) allowing continuous template-dependent replication of said target nucleic acid; and

c) sequencing said target nucleic acid by detecting in real time the individual incorporation of said first nucleotide during template-dependent replication by monitoring fluorescence emission resulting from said first label.

2 . The method of claim 1 , wherein said mixture in solution phase further comprises an activating enzyme that renders said first label fluorescent.

3 . The method of claim 2 , wherein said activating enzyme is an alkaline phosphatase, acid phosphatase, galactosidase, horseradish peroxidase, phosphodiesterase, phosphotriesterase, pyruvate kinase, lactic dehydrogenase, maltose phosphorylase, glucose oxidase, lipase, or combination thereof.

4 . The method of claim 1 , wherein said first label is photobleached after step (c).

5 . The method of claim 1 , wherein said first label is a phosphate label that is cleaved from said first nucleotide during replication.

6 . The method of claim 1 , wherein said mixture of nucleotides further comprises a second nucleotide comprising a second label that is substantially non-fluorescent until incorporation of said second nucleotide into said nucleic acid based on complementarity to said target nucleic acid.

7 . The method of claim 6 , wherein said mixture of nucleotides further comprises a third nucleotide comprising a third label that is substantially non-fluorescent until incorporation of said third nucleotide into said nucleic acid based on complementarity to said target nucleic acid.

8 . The method of claim 7 , wherein said mixture of nucleotides further comprises a fourth nucleotide comprising a fourth label that is substantially non-fluorescent until incorporation of said fourth nucleotide into said nucleic acid based on complementarity to said target nucleic acid.

9 . The method of claim 1 , further comprising repeating steps (b)-(c) at least once.

10 . The method of claim 1 , wherein said mixture in a solution phase has a volume of 0.0001 fL-1000 fL.

11 . The method of claim 1 , wherein said nucleic acid replicating catalyst is DNA polymerase, RNA polymerase, ligase, reverse transcriptase, or RNA-dependent RNA polymerase.

12 . The method of claim 1 , wherein said target nucleic acid is DNA, and said mixture in solution phase further comprises a primer.

13 . The method of claim 1 , wherein said target nucleic acid is RNA.

14 . The method of claim 1 , wherein steps (a)-(c) are repeated to obtain the sequence for 10, 25, 100, 300, 1000, or 10,000 base pairs of said target nucleic acid.

15 . The method of claim 1 , wherein said sequencing occurs continuously.

16 . The method of claim 1 , wherein said nucleic acid is immobilized on a surface of said microreactor.

17 . The method of claim 1 , wherein said nucleic acid is immobilized on a bead disposed in said microreactor.

18 . The method of claim 2 , wherein said activating enzyme is immobilized on a surface of said microreactor.

19 . The method of claim 2 , wherein said nucleic acid is immobilized on a bead disposed in said microreactor.

20 . A compound having the formula:

Base-Sugar-Phosphate-[Self-reacting Component],

where Base is a nucleotide base, Sugar is selected from the group consisting of ribose, 2′-deoxyribose, 2′-O-methyl-ribose, ribose comprising a methylene connecting the 2′ oxygen and 4′ carbon, glycerol, 2-methyl morpholine, or threose, Phosphate is a polyphosphate, and Self-reacting Component is a moiety that undergoes an intramolecular reaction upon cleavage of the phosphate to which it is connected to form a fluorophore.

21 . The compound of claim 20 , wherein Sugar is ribose or 2′-deoxyribose.

22 . The compound of claim 20 , wherein Base is cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil.

23 . The compound of claim 20 , wherein Phosphate is a triphosphate.

24 . The compound of claim 20 , wherein [Self-reacting Component] comprises a self-immolative linker.

25 . The compound of claim 20 , wherein [Self-reacting Component] comprises a moiety that undergoes an intramolecular reaction to form a fluorophore upon removal of the phosphate.

26 . The compound of claim 20 , having the formula:

wherein Q is H, OH, or OMe, n is an integer from 1 to 4; R 1 is cytosine, guanine, adenine, thymine, or uracil; L is a self-immolative linker; and R 2 is a fluorophore bound to said linker via an amine group.

27 . The compound of claim 24 , wherein said self-immolative linker is

wherein R is Phosphate; and X—NH is a fluorophore bound to said linker via an amine group.

28 . The compound of claim 27 , wherein X—NH has the formula

wherein each of R 1 -R 11 is independently selected from hydrogen, halogen, sulfonate, carboxy, C 1-6 acyl, or C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthio, a C 1-6 alkyl group interrupted with one or more heteroatoms, C 1-6 haloalkyl group, C 3-6 cycloalkyl, carboxy substituted C 1-6 alkyl, carboxy substituted C 1-6 alkoxy, carboxy substituted C 1-6 alkylthio, C 6-10 aryl, C 4-9 heteroaryl, nitro, sulfonyl substituted C 1-6 alkyl, or hydroxyl, and each Z is independently C 1-6 acyl, C 1-6 alkyl, sulfonyl, a C 1-6 alkyl group interrupted with one or more heteroatoms, C 1-6 haloalkyl group, C 3-6 cycloalkyl, carboxy substituted C 1-6 alkyl, or sulfonyl substituted C 1-6 alkyl.

29 . The compound of claim 25 , having the formula:

where Q is H, OH, or OMe, n is an integer from 1 to 4; R 1 is cytosine, guanine, adenine, thymine, or uracil; and R 2 is said moiety that undergoes an intramolecular reaction to form a fluorophore upon removal of the phosphate.

30 . The compound of claim 25 , wherein said moiety that undergoes an intramolecular reaction to form a fluorophore upon removal of the phosphate has the formula:

wherein each R is independently H or C 1-6 alkyl, or both R groups together are C 2-5 alkylene.

31 . A compound having the formula:

where R is a nucleotide base, Q is H, OH, or OMe, n is an integer from 1 to 4, and R 1 -R 10 are independently selected from hydrogen, halogen, sulfonate, carboxy, C 1-6 acyl, or C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthio, a C 1-6 alkyl group interrupted with one or more heteroatoms, C 1-6 haloalkyl group, C 3-6 cycloalkyl, carboxy substituted C 1-6 alkyl, carboxy substituted C 1-6 alkoxy, carboxy substituted C 1-6 alkylthio, C 6-10 aryl, C 4-9 heteroaryl, nitro, sulfonyl substituted C 1-6 alkyl, or hydroxyl, and X is C 1-6 acyl, C 1-6 alkyl, sulfonyl, a C 1-6 alkyl group interrupted with one or more heteroatoms, C 1-6 haloalkyl group, C 3-6 cycloalkyl, carboxy substituted C 1-6 alkyl, or sulfonyl substituted C 1-6 alkyl, wherein when R 1 -R 10 are H, X is not ethyl.

The compound of claim 31 , having the formula:

32 . The compound of claim 31 , wherein R is cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil.

33 . A compound having the formula:

wherein R is a nucleotide base, Q is H, OH, or OMe, n is an integer from 1 to 4, and R 1 -R 10 are independently selected from hydrogen, halogen, sulfonate, carboxy, C 1-6 acyl, or C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthio, a C 1-6 alkyl group interrupted with one or more heteroatoms, C 1-6 haloalkyl group, C 3-6 cycloalkyl, carboxy substituted C 1-6 alkyl, carboxy substituted C 1-6 alkoxy, carboxy substituted C 1-6 alkylthio, C 6-10 aryl, C 4-9 heteroaryl, nitro, sulfonyl substituted C 1-6 alkyl, or hydroxyl, and X is C 1-6 acyl, C 1-6 alkyl, sulfonyl, a C 1-6 alkyl group interrupted with one or more heteroatoms, C 1-6 haloalkyl group, C 3-6 cycloalkyl, carboxy substituted C 1-6 alkyl, or sulfonyl substituted C 1-6 alkyl.

34 . The compound of claim 33 , wherein R is cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil.

35 . The compound of claim 33 , having the formula: