Real-time sequencing methods and systems
The present invention is generally directed to compositions, methods, and systems for performing single-molecule, real-time analysis of a variety of different biological reactions. The ability to analyze such reactions provides an opportunity to study those reactions as well as to potentially identify factors and/or approaches for impacting such reactions, e.g., to either enhance or inhibit such reactions. In certain preferred embodiments, RNA templates are used in single-molecule real-time sequencing reactions.
1. A method of sequencing an mRNA transcript, the method comprising:
a) circularizing the mRNA transcript and, after the circularizing, providing a complex comprising the mRNA transcript and a sequencing engine, wherein the complex is immobilized in an optical confinement;
b) introducing a reaction mixture to the optical confinement, wherein the reaction mixture comprises a set of labeled nucleotides;
c) performing a sequencing-by-synthesis reaction;
d) detecting a sequence of incorporation of the labeled nucleotides into a nascent polynucleotide complementary to the mRNA transcript; and
e) determining by complementarity the sequence of the mRNA transcript based upon the sequence of incorporation of nucleotides into the nascent polynucleotide.
2. The method of claim 1 , wherein the sequencing engine is an RNA-dependent polymerase or a reverse transcriptase.
3. The method of claim 1 , wherein the sequencing engine is bound, directly or indirectly, to a surface of the optical confinement.
4. The method of claim 1 , wherein the mRNA transcript is bound, directly or indirectly, to a surface of the optical confinement.
5. The method of claim 4 , wherein the mRNA transcript is hybridized to an oligonucleotide bound to a surface of the optical confinement.
6. The method of claim 5 , wherein the mRNA transcript is hybridized to a sequence-specific region or a poly-T region of the oligonucleotide.
7. The method of claim 1 , wherein the sequencing engine processes the mRNA transcript multiple times during the sequencing-by-synthesis reaction.
8. A composition comprising an RNA-dependent polymerase bound to an RNA template in an optical confinement, and a plurality of differentially-labeled phospholinked nucleotide analogs, wherein the differentially-labeled phospholinked nucleotide analogs are unincorporatable into a nascent polynucleotide strand during template-directed synthesis by the RNA-dependent polymerase.
9. The composition of claim 8 , further comprising unlabeled phospholinked nucleotide analogs that are incorporatable into a nascent polynucleotide strand during template-directed synthesis by the RNA-dependent polymerase.
10. The composition of claim 8 , wherein the RNA-dependent polymerase is a reverse transcriptase.
11. The composition of claim 10 , wherein the reverse transcriptase is HIV reverse transcriptase.
12. The composition of claim 8 , wherein the RNA-dependent polymerase is an RNA-dependent RNA polymerase.
13. The composition of claim 12 , wherein the RNA-dependent RNA polymerase is phi6 polymerase.
14. The composition of claim 8 , wherein the optical confinement is a zero mode waveguide.
15. A system for RNA sequencing, comprising:
a) a substrate comprising a reaction site within an optical confinement, wherein a single RNA-dependent polymerase is immobilized at the reaction site and is further bound to a single RNA template molecule, wherein the RNA template molecule is a circular RNA template molecule;
b) a reaction mixture in contact with the reactions site comprising differentially labeled nucleotide analogs capable of incorporation into a nascent strand during template-directed polymerization by the single RNA-dependent polymerase on the single RNA template molecule;
c) an illumination source that provides excitation illumination;
d) an optical detector;
e) an optical train that guides the illumination from the excitation illumination source to the reaction site and guides emission signals from the reaction site to an optical detector; and
f) a recording device to record the emission signals detected by the optical detector.
16. A method of determining a nucleotide position in an RNA template, the method comprising:
a) providing an immobilized complex comprising a single RNA-dependent polymerase and a single RNA template molecule;
b) contacting the immobilized complex with a reaction mixture that comprises a set of differentially labeled nucleotides and Ca 2+ ions, and wherein the reaction mixture is deficient in Mg 2+ ions to prevent incorporation of the differentially labeled nucleotides;
c) binding one of the differentially labeled nucleotides to the immobilized complex to form a bound labeled nucleotide;
d) detecting an emission signal from the bound labeled nucleotide;
e) based upon the emission signal, determining a nucleobase in the bound labeled nucleotide and, by complementarity, an identity of the nucleotide position in the RNA template.
17. The method of claim 16 , further comprising removing the Ca 2+ ions and adding Mg 2+ ions to promote incorporation of the bound labeled nucleotide into a nascent strand complementary to the RNA template.
18. The method of claim 16 , wherein the method is repeated to promote incorporation of multiple of the differentially labeled nucleotides into the nascent strand complementary to the RNA template.