Method and system for rapid biomolecular recognition of amino acids and protein sequencing
First Claim
1. A method for specifically detecting a primary amino acid in a sample, said method comprising:
- contacting said primary amino acid to be detected with a plurality of aminoacyl tRNA synthetases, wherein each member synthetase of said plurality differs from other member synthetases of said plurality according to the cognate primary amino acids thereof and wherein said plurality of tRNA synthetases comprises an aminoacyl tRNA synthetase cognate to said primary amino acid to be detected, and wherein said contacting is under reaction conditions capable of forming a product with said primary amino acid to be detected, wherein said product is selected from the group consisting of the aminoacyl-tRNA synthetase;
amino acid AMP complex of said primary amino acid to be detected, inorganic pyrophosphate, the aminoacyl-tRNA corresponding to said primary amino acid to be detected, and AMP;
thereby forming said product; and
specifically detecting said product.
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Abstract
Methods, compositions, kits, and apparatus are provided wherein the aminoacyl-tRNA synthetase system is used to analyze amino acids. The method allows very small devices for quantitative or semi-quantitative analysis of the amino acids in samples or in sequential or complete proteolytic digestions. The methods can be readily applied to the detection and/or quantitation of one or more primary amino acids by using cognate aminoacyl-tRNA synthetase and cognate tRNA. The basis of the method is that each of the 20 synthetases and/or a tRNA specific for a different amino acid is separated spatially or differentially labeled. The reactions catalyzed by all 20 synthetases may be monitored simultaneously, or nearly simultaneously, or in parallel. Each separately positioned synthetase or tRNA will signal its cognate amino acid. The synthetase reactions can be monitored using continuous spectroscopic assays. Alternatively, since elongation factor Tu:GTP (EF-Tu:GTP) specifically binds all AA-tRNAs, the aminoacylation reactions catalyzed by the synthetases can be monitored using ligand assays. Microarrays and microsensors for amino acid analysis are provided. Additionally, amino acid analysis devices are integrated with protease digestions to produce miniaturized enzymatic sequenators capable of generating either N- or C-terminal sequence and composition data for a protein or peptide. The possibility of parallel processing of many samples in an automated manner is discussed.
160 Citations
62 Claims
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1. A method for specifically detecting a primary amino acid in a sample, said method comprising:
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contacting said primary amino acid to be detected with a plurality of aminoacyl tRNA synthetases, wherein each member synthetase of said plurality differs from other member synthetases of said plurality according to the cognate primary amino acids thereof and wherein said plurality of tRNA synthetases comprises an aminoacyl tRNA synthetase cognate to said primary amino acid to be detected, and wherein said contacting is under reaction conditions capable of forming a product with said primary amino acid to be detected, wherein said product is selected from the group consisting of the aminoacyl-tRNA synthetase;
amino acid AMP complex of said primary amino acid to be detected, inorganic pyrophosphate, the aminoacyl-tRNA corresponding to said primary amino acid to be detected, and AMP;
thereby forming said product; and
specifically detecting said product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)
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61. A method for specifically detecting a primary amino acid in a sample, said method comprising:
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contacting said primary amino acid with an aminoacyl tRNA synthetase cognate to the primary amino acid to form a product, and specifically detecting product, whereby said primary amino acid is specifically detected and wherein said sample is selected from the group consisting of cerebrospinal fluids, fermentation broths, proteolytic digests, cell culture media, blood, or serum.
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62. A method for specifically detecting a primary amino acid in a sample, said method comprising:
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contacting said primary amino acid with an aminoacyl tRNA synthetase and tRNA cognate to the primary amino acid to form a product to form an aminoacyl tRNA of the primary amino acid; and
contacting said aminoacyl tRNA with an elongation factor binary complex with GTP or a GTP analog to form a ternary complex;
and detecting said ternary complex.
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Specification