SINGLE-MOLECULE REAL-TIME ANALYSIS OF PROTEIN SYNTHESIS

• US 20100317116A1
• Filed: 06/11/2010
• Published: 12/16/2010
• Est. Priority Date: 06/12/2009
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Flusberg, Benjamin, Schadt, Eric, Turner, Stephen, Korlach, Jonas
• Granted Patent: US 8,609,421 B2
• Time in Patent Office: Days
• US Class Current: 436/56
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/6818 Sequencing of polypeptides
• CPC Class Codes: Y10T 436/13 Tracers or tags
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention is generally directed to compositions, methods, and systems for performing single-molecule, real-time analysis of analytical reactions in which protein synthesis is occurring. 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, inhibit, or otherwise affect such reactions including, but not limited to, affecting the reaction rate, processivity, fidelity, duration, and the like.

Claims

1. A method of determining a sequence of amino acids encoded by a target mRNA molecule, comprising:

a) providing a reaction mixture comprising the target mRNA molecule, a ribosome complex comprising fMet-tRNA^fMet in the P site, and a plurality of types of labeled aminoacyl-tRNAs free in solution, wherein the ribosome and/or the target mRNA molecule is immobilized upon a support such that an observation volume contains no more than one ribosome and/or mRNA molecule, and further wherein the ribosome complex does not comprise a detectable label or a quenching group;

b) initiating a processive translation of the mRNA molecule by the ribosome complex;

c) during said processive translation, sequentially and optically detecting association of the ribosome complex with at least a first labeled aminoacyl-tRNA and a second labeled aminoacyl-tRNA, where said association results in an incorporation of a first amino acid from the first labeled aminoacyl-tRNA and a second amino acid from the second labeled aminoacyl-tRNA into a nascent polypeptide chain; and

d) identifying the first amino acid and the second amino acid, thereby determining a sequence of amino acids encoded by the target mRNA molecule.

2. The method of claim 1, wherein at least 4-20 labeled aminoacyl-tRNAs are present in the reaction mixture.

3. The method of claim 1, wherein each of the plurality of types of labeled aminoacyl-tRNAs each comprise a different type of FRET acceptor.

4. The method of claim 3, wherein the reaction mixture further comprises EF-Tu comprising a FRET donor that excites each different type of FRET acceptor.

5. The method of claim 3, wherein the reaction mixture further comprises EF-G comprising a FRET donor that excites each different type of FRET acceptor.

6. The method of claim 3, wherein the reaction mixture further comprises GTP comprising a FRET donor that excites each different type of FRET acceptor.

7. The method of claim 1, wherein the reaction mixture further comprises EF-Tu comprising a quencher that quenches signal emitted from the labeled aminoacyl-tRNAs.

8. The method of claim 1, wherein the labeled aminoacyl-tRNAs each comprise a different detectable label that is not a FRET label.

9. The method of claim 8, wherein the reaction mixture further comprises at least one of the group consisting of detectably labeled EF-Tu, detectably labeled EF-G, and detectably labeled GTP.

10. The method of claim 9, wherein none of the detectably labeled EF-Tu, the detectably labeled EF-G, and the detectably labeled GTP comprises a FRET label.

11. The method of claim 1, wherein the target mRNA molecule is a circular mRNA molecule or a concatemer mRNA molecule.

16. The method of claim 1, wherein each of the plurality of types of labeled aminoacyl-tRNAs comprises (i) a different amino acid and a label that distinguishes the different amino acid from other amino acids in others of the plurality of types of labeled aminoacyl-tRNAs, and (ii) an anticodon that is complementary to a codon that may be present in the target mRNA molecule.

12-15. -15. (canceled)

17. A polypeptide sequencing method, comprising:

a) subjecting a target RNA molecule to a polypeptide synthesis reaction to yield a nascent polypeptide that is encoded by the target RNA molecule in the presence of a ribosome complex and a plurality of differentially labeled aminoacyl-tRNAs, wherein each type of differentially labeled aminoacyl-tRNA comprises

(i) a detectable label that is optically distinguishable from every other detectable label in every other type of differentially labeled aminoacyl-tRNA, and

(ii) an amino acid portion that is different from that of every other type of differentially labeled aminoacyl-tRNA,

wherein the polypeptide synthesis reaction processes an identical sequence of nucleotides in the target RNA molecule more than once;

b) detecting each type of said aminoacyl-tRNA individually by virtue of the detectable label comprised therein, wherein said detecting comprises exposing said polypeptide synthesis reaction to excitation radiation having a wavelength that is within an absorption spectrum of the detectable label; and

c) recording an order of each type of said aminoacyl-tRNA detected in step b, wherein the order is a sequence of the polypeptide synthesized in the polypeptide synthesis reaction.

18. The method of claim 17, wherein the target RNA molecule is a circular RNA molecule.

19. The method of claim 17, wherein the target RNA molecule comprises a sequence of nucleotides that is present multiple times within the target RNA molecule.

22. The method of claim 17, wherein the polypeptide synthesis reaction processes a sequence of nucleotides in the target RNA molecule multiple times.

23. The method of claim 17, wherein the ribosome complex is attached to a support and is optically resolvable from any other ribosome also attached to the support.

25. The method of claim 17, wherein the detecting and recording is performed in real time during the polypeptide synthesis reaction.

20-21. -21. (canceled)

24. (canceled)

26-27. -27. (canceled)

28. A composition comprising:

a) an optical confinement;

b) a ribosome in the optical confinement, wherein the ribosome does not comprise a label or quencher;

c) an mRNA bound to the ribosome;

d) a labeled aminoacyl-tRNA in association with the ribosome and the mRNA, wherein the labeled aminoacyl-tRNA comprises a first detectable label; and

e) an elongation factor complex comprising a second detectable label that has a different emission spectrum than an emission spectrum from the first detectable label.

29-37. -37. (canceled)

38. A method of monitoring the impact of an agent on translation of an RNA molecule, comprising:

a) subjecting a first copy of the RNA molecule to a first polypeptide synthesis reaction in the absence of the agent to generate a first set of reaction characteristics;

b) subjecting a second copy of the RNA molecule to a second polypeptide synthesis reaction in the presence of the agent to generate a second set of reaction characteristics; and

c) analyzing the first and second sets of reaction characteristics generated in step a and step b to identify the impact of the agent on translation of the RNA molecule.

49. The method of claim 38, wherein the first polypeptide synthesis reaction and the second polypeptide synthesis reaction comprise:

a) a set of labeled aminoacyl-tRNAs, wherein each labeled aminoacyl-tRNA comprises a FRET acceptor that specifies a cognate amino acid of the labeled aminoacyl-tRNA; and

b) a labeled elongation factor complex comprising a FRET donor that excites the FRET acceptor, wherein the FRET donor is linked to a component of the elongation factor complex selected from EF-Tu, EF-G, and GTP.

39-48. -48. (canceled)

50-54. -54. (canceled)

55. A method of identifying a protein encoded by a single mRNA molecule with no prior knowledge of the nucleotide sequence of the coding region of the single mRNA molecule, said method comprising:

a) providing a reaction mixture comprising the single mRNA molecule, a ribosome complex, and a plurality of types of labeled aminoacyl-tRNAs free in solution, wherein the ribosome complex does not comprise a detectable label or a quenching group;

b) initiating translation of the single mRNA molecule by the ribosome complex;

c) during said translation, sequentially and optically detecting incorporation of a set of amino acids into a nascent polypeptide chain, wherein the set of amino acids comprises amino acids from at least a portion of the plurality of labeled aminoacyl-tRNAs, wherein the polypeptide chain is at least a portion of the protein; and

d) analyzing a sequence of the incorporation of the set of amino acids to generate sequence data for the nascent polypeptide chain; and

e) using the sequence data for the polypeptide chain to identify the protein.

56. The method of claim 55, wherein the reaction mixture further comprises a plurality of types of unlabeled aminoacyl-tRNAs free in solution that are able to be incorporated into the nascent polypeptide chain, and further wherein the sequence data generated provides information for only a portion of the set of amino acids incorporated.

57-62. -62. (canceled)