Ribosome-mediated incorporation of peptides and peptidomimetics

Patent metadata

Field	Value
Publication number	US-12264318-B2
Application number	US-202217813400-A
Country	US
Kind code	B2
Filing date	Jul 19, 2022
Priority date	Jan 23, 2015
Publication date	Apr 1, 2025
Grant date	Apr 1, 2025

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.

First claim

We claim:

1. An Escherichia coli ( E. coli ) bacterial ribosome comprising a modified 23S ribosomal RNA sequence comprising modifications as compared to a wild-type counterpart, wherein the modifications consist of a first modified region (Region 1) and a second modified region (Region 2);
(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;
wherein the wild-type counterpart DNA sequence of the first unmodified region (Region 1) consists of the sequence GAAAGAC from position 2057 to 2063;
wherein the wild-type counterpart DNA sequence of the second unmodified region (Region 2) consists of the sequence GAUGUC from position 2502-2507; and
wherein the 23S ribosomal RNA is expressed from an engineered rrnB operon.

2. The genetically modified ribosome of claim 1 , wherein the Region 1 and Region 2 sequences of the modified 23S rRNA are, respectively, UGCGUGG and ACGAAG.

3. The genetically modified ribosome of claim 1 , wherein Region 1 and Region 2 sequences of the modified 23S rRNA are, respectively, UGCGUGG and CGCACG.

4. The genetically modified ribosome of claim 1 , wherein Region 1 and Region 2 sequences of the modified 23S rRNA are, respectively, UGCGUGG and CUAUGU.

5. The genetically modified ribosome of claim 1 , wherein Region 1 and Region 2 sequences of the modified 23S rRNA are, respectively, UGCGUGG and CGCAAU.

6. The genetically modified ribosome of claim 1 , wherein Region 1 and Region 2 sequences of the modified 23S rRNA are respectively, UGCGUGG and CUACAG.

7. The genetically modified ribosome of claim 1 , wherein Region 1 and Region 2 sequences of the modified 23S rRNA are, respectively, AGCGUGA and CUGCGU.

8. The genetically modified ribosome of claim 1 , wherein Region 1 and Region 2 sequences of the modified 23S rRNA are, respectively, AGCGUGA and UGGCAG.

9. The genetically modified ribosome of claim 1 , wherein Region 1 and Region 2 sequences of the modified 23S rRNA are, respectively, AGCGUGA and AUCAGG.

10. The genetically modified ribosome of claim 1 , wherein Region 1 and Region 2 sequences of the modified 23S rRNA are, respectively, AGCGUGA and AUCCGA.

11. A composition comprising the modified bacterial ribosome of claim 1 .

12. The composition of claim 11 , comprising a bacterial extract.

13. The composition of claim 11 , comprising an E. coli bacterial extract.

14. The composition of claim 11 , comprising a cell-free translation system.

15. An engineered cell comprising the modified bacterial ribosome of claim 1 .

16. The engineered cell of claim 15 , comprising an E. coli cell.

Assignees

• Hecht Sidney
• Dedkova Larisa Liza
• Maini Rumit
• Roy Chowdhury Sandipan
• Paul Rakesh
• Univ Arizona State

Inventors

• Hecht Sidney
• Dedkova Larisa (Liza)
• Maini Rumit
• Roy Chowdhury Sandipan
• Paul Rakesh

Classifications

• C12Y105/01003

  Dihydrofolate reductase (1.5.1.3) · CPC title

• C12N9/003

  Dihydrofolate reductase [DHFR] (1.5.1.3) · CPC title

• C07D277/593

  Z being doubly bound oxygen or doubly bound nitrogen, which nitrogen is part of a possibly substituted oximino radical · CPC title

• C07D263/34

  with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms · CPC title

• C12N15/11 Primary

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title