MODIFIED NUCLEOTIDE TRIPHOSPHATES FOR MOLECULAR ELECTRONIC SENSORS
In various embodiments, a signal enhancement process for single molecule molecular sensors is disclosed along with new modified dNTPs. In general, the enhancement process comprises providing a molecular electronic sensor comprising a polymerase and providing a nucleotide template and modified dNTPs in appropriate buffers to the polymerase, wherein incorporation of the modified dNTPs during action of the polymerase enzyme provides an enhanced electrical signal or improved signal-to-noise ratio for the incorporation event relative to the performance with corresponding native dNTPs.
1 . A modified nucleotide comprising a structure represented by compound [16],
wherein:
Nuc is a DNA base selected from A, T, C, and G;
Y is selected from O, S, B or I;
n is an integer from 2 to 5; and
R 1 is selected from:
wherein n=1 to 100,
wherein said modified nucleotide is incorporated by DNA polymerase in replication of a DNA template during DNA sequencing.
2 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 3; and R 1 is:
3 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 3; and R 1 is:
4 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 3; and R 1 is:
5 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 3; and R 1 is:
6 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 3; and R 1 is:
7 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 5; and R 1 is:
8 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 5; and R 1 is:
9 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 5; and R 1 is:
10 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 5; and R 1 is:
11 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 5; and R 1 is:
12 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 5; and R 1 is:
13 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is adenosine; n is 3; and R 1 is:
14 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is cytosine; n is 3; and R 1 is:
15 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is adenosine; n is 3; and R 1 is:
wherein n=1 to 100.
16 . The modified nucleotide of claim 1 , wherein Y is O; Nuc is adenosine; n is 3; and R 1 is:
17 . A modified nucleotide comprising a structure represented by compound [18],
wherein:
Nuc is a DNA base selected from A, T, C, and G;
Y is selected from OH, SH, or BH 3 ;
n is an integer from 2 to 5;
R 3 is selected from H or halogen;
R 1 is selected from H, linear or branched C 1 -C 5 alkyl, C 3 -C 8 cycloalkyl, or aryl, optionally substituted with halogen, Me or OMe, or —(CH 2 CH 2 O) x Me wherein x is an integer 1 to 20;
L 2 is selected from —(CH 2 ) q — wherein q is an integer from 1 to 10, —CH 2 CH 2 (OCH 2 CH 2 ) y — (wherein y is an integer from 1 to about 8), —(CH 2 ) q —O—(CH 2 CH 2 O) y —CH 2 — (wherein q is an integer from 1 to about 10 and y is an integer from 1 to about 8), —CO(CH 2 ) r — wherein r is an integer from 1 to about 10), —COCH 2 CH 2 (OCH 2 CH 2 ) z — wherein z is an integer from 1 to 6, —COCH 2 CH 2 CONH(CH 2 ) m — wherein m is an integer from 1 to 6, —COCH 2 CH 2 CONH(CH 2 CH 2 O) p CH 2 CH 2 — wherein p is an integer from 1 to 6, 1,4-benzenediyl, 1,3-benzenediyl, or 1,2-benzenediyl, with carbon atoms optionally and independently substituted with halogen, Me, Et, OH, OMe, or CF 3 , or,
and
R 4 and R 5 are independently selected from H, phenyl,
wherein n=1 to 100.
18 . The modified nucleotide of claim 17 , wherein: Nuc is A, T, G, or C; Y is OH; n=3 or 5; R 1 is H; R 3 is H; L 2 is —(CH 2 ) 4 —O—(CH 2 CH 2 O) 8 —CH 2 —, and R 4 is phenyl,
19 . A method of enhancing an electrical signal generated from a biosensor, the method comprising:
(a) providing a biosensor comprising source and drain electrodes and a polymerase bonded to a bridge molecule bridging the electrodes to complete an electrical circuit;
(b) placing a nucleotide template to be sequenced in communication with the polymerase;
(c) placing a modified dNTP in communication with the polymerase; and
(d) transcribing the nucleotide template by the polymerase,
wherein transcribing comprises incorporating the modified dNTP by the polymerase, and
wherein incorporating the modified dNTP results in an enhanced electrical signal compared to incorporating the corresponding non-modified dNTP.
20 . The method of claim 19 , wherein the modified dNTP comprises any one of the modified nucleotides of claims 1 - 18 .
21 . The method of claim 19 , wherein the enhanced electrical signal distinguishes between A, G, C, and T in the nucleotide template.
22 . The method of claim 19 , wherein the enhanced electrical signal is unique for incorporation of each of a modified dATP, a modified dGTP, a modified dTTP, and a modified dCTP.
23 . A method of transcribing a nucleotide template, the method comprising:
(a) providing a polymerase capable of transcribing the nucleotide template;
(b) placing the nucleotide template in communication with the polymerase;
(c) placing modified dNTPs in communication with the polymerase; and
(d) transcribing the nucleotide template with the polymerase by incorporating the modified dNTPs,
wherein the modified dNTPs comprise any one of the modified nucleotides of claims 1 - 18 .