Charge-tagged nucleotides and methods of use thereof
Provided is a method including detecting an incorporation of a labelled nucleotide into a nascent polynucleotide strand complementary to a template polynucleotide strand by a polymerase, wherein the polymerase is tethered to a solid support conductive channel by a tether and the labelled nucleotides is a compound of Formula I:
1. A method comprising:
detecting an incorporation of a labelled nucleotide into a nascent polynucleotide strand complementary to a template polynucleotide strand by a polymerase, wherein the polymerase is tethered to a solid support conductive channel by a tether,
the labelled nucleotide is a compound of Formula I
wherein n is an integer from 3 to 10,
m is an integer from 1 to 10,
t is an integer from 0 to 50,
X 1 is a direct bond, a C 1 -C 10 alkyl, a C 1 -C 10 oxaalkyl, a C 1 -C 10 thiaalkyl, or a C 1 -C 10 azaalkyl,
X 2 is C 1 -C 20 alkyl wherein optionally one or more individual CH 2 residue is replaced with one or more of a peptide bond and (—O—CH 2 —CH 2 -) a wherein a is an integer from 1 to 24,
X 3 is a direct bond or an oligonucleotide wherein the oligonucleotide hybridizes to an acceptor region of the tether when the label is in proximity to the conductive channel,
F 1 is selected from a fluorophore and a direct bond and F 2 is absent or a fluorophore,
A is
or an amide bond, and
Y is selected from
q is 1, and
B comprises a dendron of z generations comprising one or more constitutional repeating unit and a plurality of end units, wherein z is an integer from 1 to 6, the constitutional end units are selected from:
wherein
p 1 is an integer from 1 to 3, wherein any one or more of the p 1 —CH 2 — groups is optionally replaced with from 1 to 3 —O—CH 2 —CH 2 — groups,
p 2 is an integer from 1 to 3, wherein any one or more of the p 2 —CH 2 — groups is optionally replaced with from 1 to 3 —O—CH 2 —CH 2 — groups, and
the end units are selected from carboxylic acid, sulfonic acid, phosphonic acid, sperminyl group, amino group, and quaternary ammonium group, and B has a charge and a charge density, and
the charge sensor is to detect the labelled nucleotide during the incorporation.
2. The method of claim 1 , wherein the charge is between about −100e and about +100e.
3. The method of claim 2 , wherein the charge density is between about −100e per cubic nanometer and about +100e per cubic nanometer.
4. The method of claim 1 , wherein the charge is between about −200e and about +200e.
5. The method of claim 4 , wherein the charge density is between about −200e per cubic nanometer and about +200e per cubic nanometer.
6. The method of claim 1 , wherein A was formed by a reaction comprising a linking reaction and the linking reaction is selecting from an azide-alkyne copper-assisted click reaction, a tetrazine-trans-cyclooctene ligation, an azide-dibenzocyclooctyne group copper-free click reaction, and a thiol-maleimide conjugation.
7. The method of claim 1 , further comprising successively incorporating a plurality of labelled nucleotides wherein the charge of each of the plurality of labelled nucleotides differs from the charge of any other of the plurality of labelled nucleotides when the Y of the each and the Y of the any other differ from each other.
8. The method of claim 7 further comprising identifying the Y of one or more labelled polynucleotide incorporated into the nascent polynucleotide strand based on the charge detected by the conductive channel.
9. The method of any claim 1 , wherein X 2 is (—O—CH 2 —CH 2 —) a wherein a is an integer from 1 to 24.
10. The method of claim 9 , wherein a is 24.
11. The method of claim 9 , wherein a is 16.
12. The method of claim 9 , wherein a is 12.
13. The method of claim 9 , wherein a is 8.
14. The method of claim 9 , wherein a is 4.
15. The method of claim 7 , wherein the charge is between about −100e and about +100e.
16. The method of claim 15 , wherein the charge density is between about −100e per cubic nanometer and about +100e per cubic nanometer.
17. The method of claim 7 , wherein the charge is between about −200e and about +200e.
18. The method of claim 17 , wherein the charge density is between about −200e per cubic nanometer and about +200e per cubic nanometer.
19. The method of claim 8 , wherein the charge is between about −100e and about +100e.
20. The method of claim 19 , wherein the charge density is between about −100e per cubic nanometer and about +100e per cubic nanometer.
21. The method of claim 8 , wherein the charge is between about −200e and about +200e.
22. The method of claim 21 , wherein the charge density is between about −200e per cubic nanometer and about +200e per cubic nanometer.