US 11,739,378 B2
Polypeptide tagged nucleotides and use thereof in nucleic acid sequencing by nanopore detection
Frank Bergmann, Iffeldorf (DE); Christoph Seidel, Weilheim (DE); Andrew Trans, Mountain View, CA (US); Dmitriy Gremyachinskiy, Sunnyvale, CA (US); Hannes Kuchelmeister, Munich (DE); and Lars Hillringhaus, Konigsdorf-Schonrain (DE)
Assigned to Roche Sequencing Solutions, Inc., Pleasanton, CA (US); and Roche Molecular Systems, Inc., Pleasanton, CA (US)
Filed by Roche Molecular Systems, Inc., Pleasanton, CA (US); and Roche Sequencing Solutions, Inc., Pleasanton, CA (US)
Filed on Mar. 15, 2021, as Appl. No. 17/201,834.
Application 17/201,834 is a continuation of application No. 15/770,922, granted, now 10,975,426, previously published as PCT/EP2016/070198, filed on Aug. 26, 2016.
Claims priority of provisional application 62/216,634, filed on Sep. 10, 2015.
Prior Publication US 2022/0090189 A1, Mar. 24, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C07H 19/10 (2006.01); C07H 19/20 (2006.01); C12Q 1/6869 (2018.01)
CPC C12Q 1/6869 (2013.01) [C07H 19/10 (2013.01); C07H 19/20 (2013.01); C12Q 2525/113 (2013.01); C12Q 2525/197 (2013.01); C12Q 2525/203 (2013.01); C12Q 2525/204 (2013.01); C12Q 2565/631 (2013.01)] 16 Claims
 
1. A method for determining the sequence of a nucleic acid comprising:
(a) providing a nanopore sequencing composition comprising: a membrane, an electrode on the cis side and the trans side of the membrane, a nanopore with its pore extending through the membrane, an electrolyte solution in contact with both electrodes, an active polymerase situated adjacent to the nanopore, and a primer strand complexed with the polymerase;
(b) contacting the nanopore sequencing composition with (i) a strand of the nucleic acid; and (ii) a set of tagged nucleotides each with a different tag, wherein each different tag causes a different blocking current and/or blocking voltage and/or has a different dwell time when it is situated in the nanopore, and the set comprises at least one compound of structural formula (I):
N-P-L-T   (I)
wherein,
N is a nucleoside;
P is an oligophosphate covalently attached to a 5′-O group of the nucleoside, wherein the oligophosphate consists of 3 to 12 phosphate groups;
L is a linker covalently attached to a terminal phosphate group of the oligophosphate; and
T is a polypeptide tag covalently attached to the linker, wherein the polypeptide has an overall charge and comprises at least one helical structure, wherein the helical structure is an α-helix comprising the sequence motif (EAAA)16-E5; and
(c) detecting the different blocking currents and/or blocking voltages and/or different dwell times of the tags over time and correlating to each of the different tagged nucleotides incorporated by the polymerase which are complimentary to the nucleic acid sequence, and thereby determining the nucleic acid sequence.