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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
First Claim
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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-tRNAfMet 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.

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