RAMAN CLUSTER TAGGED MOLECULES FOR BIOLOGICAL IMAGING

• US 20160024570A1
• Filed: 03/14/2014
• Published: 01/28/2016
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: JU, Jingyue, KUMAR, Shiv, PALLA, Mirk, RUSSO, James J.
• Granted Patent: US 10,648,026 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer
• CPC Class Codes: G01N 21/65 Raman scattering
• CPC Class Codes: G01N 21/658 enhancement Raman, e.g. surface plasmons

Abstract

This invention provides nucleoside polyphosphate analogs each of which comprises a tag comprising a plurality of Raman-scattering moieties; compounds comprising said nucleoside polyphosphate analogs; and methods for determining the sequence of a single-stranded DNA or RNA using said nucleoside polyphosphate analogs. This invention also provides methods for detecting the interaction of a plurality of predetermined compounds, at least one of which having attached thereto a tag comprising a plurality of Raman-scattering moieties.

Claims

1. A nucleoside polyphosphate analog having the structure:

2. The nucleoside polyphosphate analog of claim 1, wherein each of the plurality of Raman-scattering moieties has a Raman spectroscopy peak with wavenumber from 2000 cm⁻

1 to 2300 cm<sup>−

1</sup>.

3. The nucleoside polyphosphate analog of claim 1, wherein each of the plurality of Raman-scattering moieties is selected from the group consisting of —

N═

N═

N, —

C≡

N, —

C≡

CH, and —

C≡

C—

CH₃.

4. The nucleoside polyphosphate analog of claim 1, wherein the tag comprising a plurality of identical Raman-scattering moieties.

5. The nucleoside polyphosphate analog of claim 1, wherein the tag comprising a plurality of different Raman-scattering moieties.

6. The nucleoside polyphosphate analog of claim 1, wherein the tag comprising 3 Raman-scattering moieties.

9. The nucleoside polyphosphate analog of claim 1, wherein the plurality of Raman-scattering moieties forms a linear tag.

10. The nucleoside polyphosphate analog of claim 1, wherein the plurality of Raman-scattering moieties forms a non-linear tag.

11. The nucleoside polyphosphate analog of claim 10, wherein the non-linear tag is a dendrimer tag.

12. The nucleoside polyphosphate analog of claim 2, wherein the tag has a Raman spectroscopy peak with wavenumber from 2125 cm⁻

1 to 2260 cm<sup>−

1</sup>.

13. The nucleoside polyphosphate analog of claim 1, wherein the linker is selected from the group consisting of O, NH, S, CH₂, amino acids, peptides, proteins, carbohydrates, polyethylene glycols of different length and molecular weights, aliphatic acids, aromatic acids, alcohols or thiol groups (substituted or unsubstituted), cyano groups, nitro groups, alkyl groups, alkenyl groups, alkynyl groups, and azido groups.

7-8. -8. (canceled)

14. A composition comprising four deoxyribonucleoside polyphosphate (dNPP) analogs, each dNPP analog having the structure:

15. A composition comprising four ribonucleoside polyphosphate (rNPP) analogs, each rNPP analog having the structure:

16-27. -27. (canceled)

28. A method for determining the sequence of a single-stranded DNA comprising:

(a) contacting the single-stranded DNA having a primer hybridized to a portion thereof with a DNA polymerase and four deoxyribonucleoside polyphosphate (dNPP) analogs under conditions permitting the DNA polymerase to catalyze incorporation onto the primer of a dNPP analog complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′

to a nucleotide residue of the single-stranded DNA hybridized to the 3′

terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein each dNPP analog has the structure;

30. The method of claim 28, wherein the polymerase is immobilized on a solid surface.

33. The method of claim 28, wherein the polymerase is immobilized such that only the tagged polyphosphate released in step (a) is within the detection range of the SERS detector.

34. The method of claim 33, wherein the polymerase is immobilized such that the tagged polyphosphate released in step (a) is within a cavity on the SERS substrate.

46. The method of claim 28, wherein the linker is selected from the group consisting of O, NH, S, CH₂, amino acids, peptides, proteins, carbohydrates, polyethylene glycols of different length and molecular weights, aliphatic acids, aromatic acids, alcohols or thiol groups (substituted or unsubstituted), cyano groups, nitro groups, alkyl groups, alkenyl groups, alkynyl groups, and azido groups.

29. A method for determining the sequence of a single-stranded RNA comprising:

(a) contacting the single-stranded RNA having a primer hybridized to a portion thereof with an RNA polymerase and four ribonucleoside polyphosphate (rNPP) analogs under conditions permitting the RNA polymerase to catalyze incorporation onto the primer of an rNPP analog complementary to a nucleotide residue of the single-stranded RNA which is immediately 5′

to a nucleotide residue of the single-stranded RNA hybridized to the 3′

terminal nucleotide residue of the primer, so as to form an RNA extension product, wherein each rNPP analog has the structure;

31. The method of claim 29, wherein the Raman spectroscopy peak is determined using surface-enhanced Raman spectroscopy (SERS).

32. The method of claim 31, wherein the polymerase is immobilized on a SERS substrate.

35-45. -45. (canceled)

47. A method for detecting interaction of a plurality of predetermined compounds comprising:

(a) contacting at least a first predetermined compound immobilized on a solid surface with a solution containing at least a second predetermined compound, wherein the second predetermined compound having attached thereto a tag comprises a plurality of Raman-scattering moieties, under conditions permitting binding of said at least first and second predetermined compounds such that only said second predetermined compound bound to said immobilized first predetermined compound is within the detection range of a detector; and

(b) performing Raman spectroscopy, wherein detection of a Raman spectroscopy peak indicating presence of the tag and binding of said at least first and second predetermined compounds, so as to thereby detecting the interaction of the plurality of predetermined compounds.

48-74. -74. (canceled)