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US 10,975,426 B2Granted
Polypeptide tagged nucleotides and use thereof in nucleic acid sequencing by nanopore detection
AssigneeRoche Sequencing Solutions, Inc.
InventorsFrank Bergmann, Christoph Seidel, Andrew Trans, Dmitriy Gremyachinskiy, Hannes Kuchelmeister, Lars Hillringhaus
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Abstract
The present disclosure relates to compositions and methods based on polypeptide-tagged nucleotide, and the use of such polypeptide-tagged nucleotides in nanopore devices and methods.
Claims13
A 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 α-helix comprising at least 2 repeats of a sequence motif, wherein the repeats are not interrupted by an amino acid residue that is non-helix-forming, and the amino acid sequence motif is selected from the group consisting of:
- EAAA, AEAA, AAEA, AAAE, DAAA, ADAA, AADA, AAAD, RAAA, ARAA, AARA, AAAR, KAAA, AKAA, AAKA, and AAAK.
6 more elements
A method of preparing a polypeptide-tagged nucleotide compound of structural formula (II)
- wherein,
- Base is selected from adenosine, cytidine, guanosine, thymidine, and uridine;
- R is selected from H and OH;
- n is from 1 to 4;
- Linker is a linker comprising a covalently bonded chain of 2 to 100 atoms;
- and
- Polypeptide is a polypeptide tag comprising an overall charge and at least one α-helix comprising at least 2 repeats of a sequence motif, wherein the repeats are not interrupted by an amino acid residue that is non-helix-forming, and the amino acid sequence motif is selected from the group consisting of:
- EAAA, AEAA, AAEA, AAAE, DAAA, ADAA, AADA, AAAD, RAAA, ARAA, AARA, AAAR, KAAA, AKAA, AAKA, and AAAK
- the method comprising:
- (a) providing (i) a nucleotide with from 3 to 12 phosphates attached to its 5′-position, wherein the terminal phosphate is coupled to a first linker forming group;
- and (ii) a polypeptide tag, wherein the polypeptide tag comprises at least one α-helix comprising at least 2 repeats of a sequence motif, wherein the repeats are not interrupted by an amino acid residue that is non-helix-forming, and the amino acid sequence motif is selected from the group consisting of:
- EAAA, AEAA, AAEA, AAAE, DAAA, ADAA, AADA, AAAD, RAAA, ARAA, AARA, AAAR, KAAA, AKAA, AAKA, and AAAK, has an overall charge, and is coupled to a second linker forming group, that is capable of reacting with the first linker forming group to form a linker;
- wherein
- the first linker forming group is selected from the compounds of structural formulas (IVa)-(XVIIa) and the second linker forming group is the corresponding reactive compound of structural formulas (IVb)-(XVIIb);
- or
- the first linker forming group is selected from the compounds of structural formulas (IVb)-(XVIIb) and the second linker forming group is the corresponding reactive compound of structural formulas (IVa)-(XVIIa);
- and
- (b) reacting the first linker forming group with the second linker forming group, thereby forming a covalent linkage between the nucleotide to the polypeptide tag.
15 more elements
Dependencies are inferred from the claim text.