Posted by: shrikantmantri | February 15, 2010

Nature Paper: Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome (DNA 2.0: A new operating system for life is created)

Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome

Heinz Neumann1,2, Kaihang Wang1,2, Lloyd Davis1, Maria Garcia-Alai1& Jason W. Chin1

  1. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK
  2. These authors contributed equally to this work.

Correspondence to: Jason W. Chin1 Correspondence and requests for materials should be addressed to J.W.C. (Email: chin@mrc-lmb.cam.ac.uk).

Nature advance online publication 14 February 2010 | 

doi:10.1038/nature08817

; Received 26 June 2009; Accepted 7 January 2010; Published online 14 February 2010

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The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision1. The Methanococcus jannaschiityrosyl-transfer-RNA synthetase–tRNACUA (MjTyrRS–tRNACUA)2,3 and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase–tRNACUA (MbPylRS–tRNACUA)4, 5, 6 orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli1, 6, 7. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase–tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome8, 9 (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates8. By creating mutually orthogonal aminoacyl-tRNA synthetase–tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids10. Because the synthetase–tRNA pairs used have been evolved to incorporate numerous unnatural amino acids1, 6, 7, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.

Source:http://www.nature.com/nature/journal/vaop/ncurrent/full/nature08817.html

Posted via email from Sharing significant bytes —(Shrikant Mantri)

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