Compositions and methods for sequencing nucleic acids

A compound having Formula I:

• or a pharmaceutically acceptable salt or hydrate thereof, wherein
• n is 1, 2, 3, 4, 5, 6, 7, 8, or 9;
• R1 and each R2 is chosen from O− and
• wherein
• i) R1 and each R2 are O−;
• or
• ii) R1 is
• and each R2 is O−;
• or
• iii) R1 is O−, one R2 is
• and any remaining R2 is independently O−, S−, BH3−, or CH3;
• R3 is a nucleotide moiety comprising a fluorescent dye F;
• R4 is H, OH, halogen (including fluorine, chlorine, bromine, and iodine), alkyl (including CH3, CH2CH3) or alkoxy (both substituted and unsubstituted) (including OCH3 and OCH2CH3);
• Y1, and Y3 are each independently chosen from O−, S−, BH3−, and CH3;
• L1 is chosen from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ester, amino, and sulfonyl;
• Q is a fluorescence quenching moiety;
• and
• B1 is chosen from adenine, cytosine, guanine, thymine, uracil, hypoxanthine, and 5-methylcytosine.

A method of determining a nucleotide base incorporated by a polymerase enzyme or enzyme complex in a nucleic acid polymerization reaction, the method comprising the steps of:

• (a) conducting a nucleic acid polymerization reaction that utilizes both 5′ to 3′ polymerization activity and 3′ to 5′ exonuclease activity of a polymerase enzyme or enzyme complex, and that results in production of a nascent strand in a template-dependent manner, wherein said reaction is conducted in the presence of
• (i) a template nucleic acid comprising a target nucleic acid sequence,
• (ii) a primer nucleic acid comprising a sequence which is complementary to a region of the template nucleic acid,
• (iii) a polymerase enzyme or an enzyme complex comprising 5′ to 3′ polymerization activity and 3′ to 5′ exonuclease activity,
• (iv) a plurality of nucleotide analogs, wherein an individual nucleotide analog of said plurality comprises at least one base-pairing moiety and at least one label moiety, said label moiety comprising a photo-detectable label;
• and
• (b) detecting the photo-detectable label, wherein said label is indicative of the identity of the base or bases present in the nucleotide analog incorporated by the enzyme or enzyme complex into the nascent strand.

A method of determining the nucleic acid sequence of a target nucleic acid sequence, the method comprising the steps of:

• (a) conducting a nucleic acid polymerization reaction in the presence of
• (i) a template nucleic acid comprising a target nucleic acid sequence,
• (ii) a primer nucleic acid comprising a sequence which is complementary to a region of the template nucleic acid,
• (iii) a polymerase enzyme or enzyme complex comprising a reaction site for incorporating a nucleotide analog in a template-dependent manner,
• (iv) a plurality of nucleotide analogs, wherein an individual nucleotide analog of said plurality comprises at least one base-pairing moiety and at least one label moiety comprising a photo-detectable label, and wherein the analog does not terminate production of a nascent strand once the analog is incorporated in a template-dependent manner by the enzyme or enzyme complex into the nascent strand, and wherein said nucleotide analog provides at least one of prolonged detection duration and prolonged interpulse duration as compared to a nucleotide analog comprising a label which is removed via the 5′ to 3′ polymerization activity of the enzyme or enzyme complex;
• and
• (b) detecting the label moiety incorporated during each successive incorporation event.