Ribosome-Mediated Incorporation of Peptides and Peptidomimetics
AssigneeArizona Board of Regents on behalf of Arizona State University
InventorsSidney Hecht, Larisa (Liza) Dedkova, Rumit Maini, Sandipan Roy Chowdhury, Rakesh Paul
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Abstract
Modified ribosomes that were selected using a dipeptidyl-puromycin aminonucleoside are used to mediate site-specific incorporation of one or more peptides and peptidomimetics into protein in a cell free translation system. In addition, new fluorescent dipeptidomimetics have been synthesized and incorporated into proteins, as well as modified proteins containing one or more non-naturally occurring dipeptides.
Claims16
A method for producing a peptide comprising one or more dipeptide or dipeptidomimetic, the method comprising:
- (a) contacting an mRNA transcript encoding the peptide with a modified ribosome in a cell-free translation system;
- wherein the modified ribosome consists of an of an Escherichia coli (E. coli) 23S ribosomal RNA (rRNA) sequence modification, the 23S rRNA sequence modification consisting of a modification in Region 1 (position 2057-2063) selected from UGCGUGG or AGCGUGA, and a modification in Region 2 (position 2502-2507);
- wherein the mRNA transcript comprises one or more stop codons (UAA, UAG, UGA) at one or more amino acid positions for incorporation of the dipeptide or dipeptidomimetic;
- wherein the cell-free translation system comprises one or more suppressor tRNAs corresponding to the one or more stop codons, the suppressor tRNA charged with the one or more dipeptide or a dipeptidomimetic;
- (b) incubating the contacted mRNA transcript with the modified ribosome in the cell-free translation system;
- and
- (c) producing the peptide comprising the one or more dipeptide or dipeptidomimetic.
An in vitro translation system comprising:
- a genetically modified bacterial ribosome, the modification consisting of an Escherichia coli (E. coli) 23S ribosomal RNA (rRNA) sequence modification, the 23S rRNA sequence modification consisting of a modification in Region 1 (position 2057-2063) selected from UGCGUGG or AGCGUGA, and a modification in Region 2 (position 2502-2507), wherein:
- (a) if the modification in Region 1 consists of UGCGUGG from position 2057-2063, the modification in Region 2 is selected from the group consisting of ACGAAG, CGCACG, CUAUGU, CGCAAU, or CUACAG from position 2502-2507;
- (b) if the modification in Region 1 consists of AGCGUGA from position 2057-2063, the modification in Region 2 is selected from the group consisting of CUGCGU, UGGCAG, AUCAGG, or AUCCGA from position 2502-2507.
A modified peptide comprising one or more dipeptide or dipeptidomimetic selected from the group consisting of:
Dependencies are inferred from the claim text.