Methods, systems, and reagents for direct RNA sequencing

• US 10,280,411 B2
• Filed: 10/26/2016
• Issued: 05/07/2019
• Est. Priority Date: 10/27/2015
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Kamtekar, Satwik, Hanes, Jeremiah, Miller, Erik
• Primary Examiner(s): Hutson, Richard G
• Application Number:
  US15/334,935
• Publication Number:
  US 20170159033A1
• Time in Patent Office: 923 Days
• Field of Search: None
• CPC Class Codes: C12M 1/42 Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)

Abstract

Provided are compositions comprising recombinant polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for sequencing RNA or RNA/DNA templates. Polymerases that topologically encircle the template nucleic acid are provided. Also provided are methods of using such polymerases to make a DNA or to sequence a template comprising RNA.

Claims

1. A reaction mixture comprising a polymerase enzyme complex comprising a polymerase enzyme and a template nucleic acid that is bound to the polymerase,

wherein the polymerase is a recombinant Φ

29-type polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO;

1 or at least 80% identical to SEQ ID NO;

2, and which recombinant polymerase comprises a) a first cysteine residue at position V19 and a second cysteine residue at position N409, b) a first cysteine residue at position D84 and a second cysteine residue at position E418, c) a first cysteine residue at position N409 and a second cysteine residue at position V568, or d) a first cysteine residue at position A83 and a second cysteine residue at position E420, wherein identification of positions is relative to SEQ ID NO;

1,

wherein the side chains of the first and second cysteine residues are covalently connected by a linker, thereby topologically encircling the template nucleic acid.

2. The reaction mixture of claim 1, wherein the template nucleic acid is circular.

3. The reaction mixture of claim 1, wherein the template nucleic acid is an mRNA.

4. The reaction mixture of claim 1, comprising a primer hybridized to the template nucleic acid.

5. The reaction mixture of claim 1, wherein the polymerase enzyme complex is immobilized on a surface.

6. The reaction mixture of claim 5, comprising sequencing reagents in contact with the surface, which sequencing reagents comprise reagents for carrying out nucleic acid synthesis including one or more labeled nucleotide analogs.

7. The reaction mixture of claim 1, comprising a phosphate-labeled nucleotide analog, wherein the polymerase incorporates the nucleotide analog into a copy nucleic acid in response to the template nucleic acid.

8. The reaction mixture of claim 1, wherein the recombinant polymerase comprises a first cysteine residue at position V19 and a second cysteine residue at position N409, wherein identification of positions is relative to SEQ ID NO:

1, wherein the side chains of the cysteine residues at positions V19 and N409 are covalently connected by the linker.

9. The reaction mixture of claim 1, wherein the recombinant polymerase comprises a first cysteine residue at position D84 and a second cysteine residue at position E418, wherein identification of positions is relative to SEQ ID NO:

1, wherein the side chains of the cysteine residues at positions D84 and E418 are covalently connected by the linker.

10. The reaction mixture of claim 1, wherein the recombinant polymerase comprises a first cysteine residue at position N409 and a second cysteine residue at position V568, wherein identification of positions is relative to SEQ ID NO:

1, wherein the side chains of the cysteine residues at positions N409 and V568 are covalently connected by the linker.

11. The reaction mixture of claim 1, wherein the recombinant polymerase comprises a first cysteine residue at position A83 and a second cysteine residue at position E420, wherein identification of positions is relative to SEQ ID NO:

1, wherein the side chains of the cysteine residues at positions A83 and E420 are covalently connected by the linker.

14. The reaction mixture of claim 1, wherein the template nucleic acid comprises RNA.

15. The reaction mixture of claim 1, wherein the template nucleic acid comprises DNA.

16. The reaction mixture of claim 1, wherein the recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

1 or at least 90% identical to SEQ ID NO;

2.

12. A method for sequencing a nucleic acid template comprising:

providing a polymerase enzyme complex comprising a polymerase enzyme, a template nucleic acid that is bound to the polymerase, and optionally a primer hybridized to the template nucleic acid, wherein the polymerase is a recombinant Φ

29-type polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO;

1 or at least 80% identical to SEQ ID NO;

2, and which recombinant polymerase comprises a) a first cysteine residue at position V19 and a second cysteine residue at position N409, b) a first cysteine residue at position D84 and a second cysteine residue at position E418, c) a first cysteine residue at position N409 and a second cysteine residue at position V568, or d) a first cysteine residue at position A83 and a second cysteine residue at position E420, wherein identification of positions is relative to SEQ ID NO;

1, wherein the side chains of the first and second cysteine residues are covalently connected by a linker, thereby topologically encircling the template nucleic acid, wherein the polymerase enzyme complex is immobilized on a surface;

adding sequencing reagents in contact with the surface comprising reagents for carrying out nucleic acid synthesis including one or more labeled nucleotide analogs; and

determining the sequential addition of nucleotide residues to a nucleic acid strand complementary to a strand of the template nucleic acid by observing the interaction of the labeled nucleotide analogs with the polymerase enzyme complex.

13. A system for sequencing nucleic acids comprising:

a chip comprising a plurality of polymerase enzyme complexes bound thereto, each polymerase enzyme complex individually optically resolvable, each polymerase enzyme complex comprising a polymerase enzyme, a template nucleic acid that is bound to the polymerase, and optionally a primer hybridized to the template nucleic acid, wherein the polymerase is a recombinant Φ

29-type polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO;

1 or at least 80% identical to SEQ ID NO;

2, and which recombinant polymerase comprises a) a first cysteine residue at position V19 and a second cysteine residue at position N409, b) a first cysteine residue at position D84 and a second cysteine residue at position E418, c) a first cysteine residue at position N409 and a second cysteine residue at position V568, or d) a first cysteine residue at position A83 and a second cysteine residue at position E420, wherein identification of positions is relative to SEQ ID NO;

1, wherein the side chains of the first and second cysteine residues are covalently connected by a linker, thereby topologically encircling the template;

sequencing reagents in contact with the surface comprising reagents for carrying out nucleic acid synthesis including one or more labeled nucleotide analogs;

an illumination system for illuminating the polymerase enzyme complexes;

an optical detection system for detecting fluorescence from the labeled nucleotide analogs while they are interacting with the polymerase enzyme complexes; and

a computer for analyzing the signals detected by the detection system to determine the sequential addition of nucleotide residues to a nucleic acid strand complementary to a strand of the template nucleic acid.