Raman cluster tagged molecules for biological imaging

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:
• wherein B is a base and is adenine, guanine, cytosine, thymine, or a derivative thereof, wherein R1 is OH, wherein R2 is H, wherein n is 1, 2, 3, or 4, wherein X is a linker, wherein the tag comprises a plurality of identical Raman-scattering moieties,
• wherein (i) the Raman spectroscopy peak of the tag on each dNPP analog is distinguishable from the Raman spectroscopy peak of the tag on each of the remaining three dNPP analogs, and (ii) each dNPP analog comprises a base which is different from the base of each of the remaining three dNPP analogs, and
• wherein the incorporation of the dNPP analog releases a tagged polyphosphate;
• (b) determining the wavenumber of the Raman spectroscopy peak of the tagged polyphosphate released in step (a), so as to thereby determine the identity of the incorporated dNPP analog and the identity of the complementary nucleotide residue in the single-stranded DNA;
• and
• (c) iteratively performing steps (a) and (b) for each nucleotide residue of the single-stranded DNA to be sequenced so as to thereby determine the sequence of the single-stranded DNA.

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:
• wherein B is a base and is adenine, guanine, cytosine, uracil, or a derivative thereof, wherein R1 is OH, wherein R2 is OH, wherein n is 1, 2, 3, or 4, wherein X is a linker, wherein the tag comprises a plurality of identical Raman-scattering moieties,
• wherein (i) the Raman spectroscopy peak of the tag on each rNPP analog is distinguishable from the Raman spectroscopy peak of the tag on each of the remaining three rNPP analogs, and (ii) each rNPP analog comprises a base which is different from the base of each of the remaining three rNPP analogs, and
• wherein the incorporation of the rNPP analog releases a tagged polyphosphate;
• (b) determining the wavenumber of the Raman spectroscopy peak of the tagged polyphosphate released in step (a), so as to thereby determine the identity of the incorporated rNPP analog and the identity of the complementary nucleotide residue in the single-stranded RNA;
• and
• (c) iteratively performing steps (a) and (b) for each nucleotide residue of the single-stranded RNA to be sequenced so as to thereby determine the sequence of the single-stranded RNA.