Synthesis and use of anti-reverse mRNA cap analogues

• US 7,074,596 B2
• Filed: 05/17/2002
• Issued: 07/11/2006
• Est. Priority Date: 03/25/2002
• Status: Active Grant

• Current Assignee: Edward Darzynkiewicz, Board of Supervisors of Louisiana State University Agricultural & Mechanical College (Louisiana State University System)
• Original Assignee: Board of Supervisors of Louisiana State University Agricultural & Mechanical College (Louisiana State University System)
• Inventors: Darzynkiewicz, Edward, Stepinski, Janusz, Rhoads, Robert E.
• Primary Examiner(s): Lilling, Herbert J.
• Application Number:
  US10/150,718
• Publication Number:
  US 20030194759A1
• Time in Patent Office: 1,516 Days
• Field of Search: 435/5, 435/6, 435/90, 435/91, 435/91.1, 435/91.51, 536/23.1, 536/23.2, 536/25.4
• US Class Current: 435/90
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

The ability to synthesize capped RNA transcripts in vitro has been of considerable value in a variety of applications. However, one-third to one-half of the caps have, until now, been incorporated in the reverse orientation. Such reverse caps impair the translation of in vitro-synthesized mRNAs. Novel cap analogues, such as P¹-3′-deoxy-7-methylguanosine-5′P³-guanosine-5′triphosphate and P¹-3′-O,7-dimethylguanosine-5′P³-guanosine-5′triphosphate, have been designed that are incapable of being incorporated into RNA in the reverse orientation. Transcripts produced with SP6 polymerase using “anti-reverse” cap analogues were of the predicted length. Analysis of the transcripts indicated that reverse caps were not formed. The in vitro translational efficiency of transcripts with the novel “anti-reverse” cap analogues was significantly higher than that of transcripts formed with conventional caps.

Claims

1. A composition comprising

2. A composition as recited in claim 1, wherein X is OH, Y is H, and n is 1.

3. A composition as recited in claim 1, wherein X is OH, Y is OCH₃, and n is 1.

4. An RNA molecule whose 5′

end incorporates a composition as recited in claim 1.

5. An RNA molecule whose 5′

end incorporates a composition as recited in claim 2.

6. An RNA molecule whose 5′

end incorporates a composition as recited in claim 3.

7. A method for synthesizing an RNA molecule as recited in claim 4 in vitro;

said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template;

in the presence an RNA polymerase;

under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy;

whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

8. A method for synthesizing an RNA molecule as recited in claim 5 in vitro;

said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template;

in the presence an RNA polymerase;

under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy;

whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

9. A method for synthesizing an RNA molecule as recited in claim 6 in vitro;

said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template;

in the presence an RNA polymerase;

under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy;

whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

10. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 4 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

11. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 5 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

12. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 6 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.