Compositions and methods for sequencing nucleic acids
Embodiments relate to methods of sequencing nucleic acids. Embodiments encompass the use of nucleotide analogs and a nucleic acid polymerase enzyme or enzyme complex comprising proofreading activity. The nucleotide analogs may become incorporated into a replicating strand and induce the proofreading activity of the polymerizing enzyme, thereby prolonging the duration of a signal associated with nucleotide incorporation, resulting in more observable sequencing events and increasing the accuracy of nucleic acid sequencing.
1. 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;
i) R 1 and each R 2 are O − ; or
ii) R 1 is
and each R 2 is O − ; or
iii) R 1 is O − , one R 2 is
and any remaining R 2 is independently O − , S − , BH 3 − , or CH 3 ;
R 3 is a nucleotide moiety comprising a fluorescent dye F and at least one non-complementary nucleotide residue;
R 4 is H, OH, halogen, alkyl (both substituted and unsubstituted), or alkoxy (both substituted and unsubstituted);
Y 1 , and Y 3 are each independently chosen from O − , S − , BH 3 − , and CH 3 ;
L 1 is chosen from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ester, amino, and sulfonyl;
Q is a fluorescence quenching moiety; and
B 1 is chosen from adenine, cytosine, guanine, thymine, uracil, hypoxanthine, and 5-methylcytosine.
2. The compound of claim 1 , wherein R 3 is chosen from:
B 2 is chosen from adenine, cytosine, guanine, thymine, uracil, hypoxanthine, and 5-methylcytosine;
X 1 is chosen from methylene; L 2 ; a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil; and groups comprising L 2 and a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
wherein L 2 is chosen from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ester, amino, and sulfonyl;
X 2 is chosen from H, CH 3 , and a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
each R 5 is independently H, OH, fluorine, or OCH 3 ; and
Y 2 is chosen from O − , S − , BH 3 − , and CH 3 .
3. The compound of claim 1 , wherein R 3 is chosen from:
B 2 is chosen from adenine, cytosine, guanine, thymine, uracil, hypoxanthine, and 5-methylcytosine;
X 1 is chosen from methylene; L 2 ; a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil; and a group comprising L 2 and a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
wherein L 2 is chosen from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ester, amino, and sulfonyl;
X 2 is chosen from H, CH 3 , and a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil; and
Y 2 is chosen from O − , S − , BH 3 − , and CH 3 .
4. The compound of claim 2 , wherein n is 1, R 1 is
and R 2 is O − .
5. The compound of claim 2 , wherein Y 1 is S − .
6. The compound of claim 2 , wherein R 3 is
7. The compound of claim 2 , wherein Y 3 is O − .