Ribosome-mediated incorporation of peptides and peptidomimetics
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.
1. An in vitro translation system comprising materials sufficient to effect translation from a messenger RNA (mRNA), comprising:
a transfer RNA (tRNA) with modified dipeptidomimetic
and
a genetically modified ribosome having a 23S ribosomal RNA (rRNA) sequence modification selected from the group consisting of one or more modification(s) selected from the following:
Region 1
2056
2057
2058
2059
2060
2061
2062
2063
2064
G
U
G
C
G
U
G
G
C
A
U
A
A
Region 2
2501
2502
2503
2504
2505
2506
2507
2508
C
U
C
G
U
G
U
G
A
U
A
A
A
A
C
G
C
C
C
G.
2. The system of claim 1 , wherein said materials comprise an E. coli S-30 extract.
3. A method for producing a modified peptide, polypeptide, or protein, comprising the step of:
providing to an in vitro translation system a transfer RNA (tRNA) with modified dipeptidomimetic
said system comprising materials sufficient to effect translation from a messenger RNA (mRNA) and containing a genetically modified ribosome having a 23 S ribosomal RNA (rRNA) sequence modification selected from the group consisting of one or more modification(s) selected from the following:
Region 1
2056
2057
2058
2059
2060
2061
2062
2063
2064
G
U
G
C
G
U
G
G
C
A
U
A
A
Region 2
2501
2502
2503
2504
2505
2506
2507
2508
C
U
C
G
U
G
U
G
A
U
A
A
A
A
C
G
C
C
C
G.
4. The method of claim 3 , wherein said materials include a mRNA encoding the protein(s) of interest and containing one or more stop codons, and one or more suppressor tRNA(s) activated with the modified dipeptidomimetic recognized by the selected ribosomes.