Method of site-specific alteration of RNA and production of encoded polypeptides

• US 5,635,377 A
• Filed: 11/18/1994
• Issued: 06/03/1997
• Est. Priority Date: 02/15/1990
• Status: Expired due to Fees

• Current Assignee: Worcester Foundation For Biomedical Research, Inc.
• Original Assignee: Worcester Foundation For Experimental Biology
• Inventors: Pederson, Thoru, Mayrand, Sandra, Agrawal, Sudhir, Zamecnik, Paul C.
• Primary Examiner(s): Patterson, Jr., Charles L.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US08/342,302
• Time in Patent Office: 928 Days
• Field of Search: 435/69.1, 435/91.3, 536/23.1, 536/29.13, 536/22.1
• US Class Current: 435/91.3
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/8233 Female-specific, e.g. pistil, ovule
• CPC Class Codes: C12N 15/8235 Fruit-specific
• CPC Class Codes: C12N 2310/3125 Methylphosphonates
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates

Abstract

A method of site-directed alteration (removal or removal followed by replacement) of selected nucleotides in an RNA molecule, as well as to mixed phosphate backbone oligonucleotides useful in the method. It further relates to a method of producing polypeptides or proteins encoded by the RNA molecule altered by the present method. Through use of the present method, site-directed cleavage of an RNA molecule is effected, followed by excision of the selected or target segment of the RNA molecule.

Claims

1. A method of site-specific cleavage of target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all of a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide including an internal segment that activates RNase H and two flanking nucleotide sequences that are unable to activate RNase H, one of the two flanking nucleotide sequences being present on either side of the internal segment, wherein the internucleoside bridging phosphate residues of the internal segments are modified phosphates that are phosphorothioates and the internucleoside bridging phosphate residues of the two flanking modified nucleotide sequences are methyl phosphonates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

2. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide including an internal segment that activates RNase H and two flanking nucleotide sequence that are unable to activate RNase H, one of the two flanking nucleotide sequences being present on either side of the internal segment, wherein the internucleoside bridging phosphate residues of the internal segment are modified phosphates that are phosphorothioates and the internucleoside bridging phosphate residues of the two flanking modified nucleotide sequences are phosphoromorpholidates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

3. A method of site-specific cleavage of target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide including an internal segment that activates RNase H and two flanking nucleotide sequences that are unable to activate RNase H, one of the two flanking nucleotide sequences being present on either side of the internal segment, wherein the internucleoside bridging phosphate residues not the internal segment are modified phosphates that are phosphorothioates and the internucleoside bridging phosphate residues of the two flanking modified nucleotide sequences are phosphoropiperazidates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target of RNA segment.

4. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining then RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide including an internal segment that activates RNase H and two flanking nucleotide sequences that are unable to activate RNase H, one of the two flanking nucleotide sequences being present on either side of the internal segment, wherein the internucleoside bridging phosphate residues of the internal segment are modified phosphates that are phosphorothioates and the internucleoside bridging phosphate residues of the two flanking modified nucleotide sequences are phosphoramidates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

5. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNA H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that activate RNase H and a 3'"'"' segment of nucleotides that does not activate RNase H, wherein the 5'"'"' segment of nucleotides comprises nucleotide phosphodiesters and wherein the 3'"'"' segment of nucleotides comprises two or more contiguous nucleotide alkylphosphonates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNAase H, thereby cleaving the target RNA segment.

6. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that activate RNase H and a 3'"'"' segment of nucleotides that do not activate RNase H, wherein the 5'"'"' segment of nucleotides comprises nucleotide phosphodiesters and wherein the 3'"'"' segment of nucleotides comprises nucleotide phosphoromorpholidates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

7. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that activate RNase H and a 3'"'"' segment of nucleotides that don not activate RNase H, wherein the 5'"'"' segment of nucleotides comprises nucleotide phosphodiesters and wherein the 3'"'"' segment of nucleotides comprises nucleotide phosphoramidates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

8. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that do not activate RNase H and a 3'"'"' segment of nucleotides that activate RNase H, wherein the 5'"'"' segment of nucleotides comprises three of more contiguous nucleotide alkylphosphonates and the 3'"'"' segment of nucleotides comprises nucleotide phosphodiesters, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

9. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that do not activate RNase H and a 3'"'"' segment of nucleotides that activate RNase H, wherein the 5'"'"' segments of nucleotides comprises nucleotide phosphoromorpholidates and the 3'"'"' segment of nucleotides comprises nucleotide phosphodiesters, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

10. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that do not activate RNase H and a 3'"'"' segment of nucleotides that activate RNase H, wherein the 5'"'"' segment of nucleotides comprises nucleotide phosphoramidates and the 3'"'"' segment of nucleotides comprises nucleotide phosphodiesters, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activating of RNase H, thereby cleaving the target RNA segment.

11. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that activate RNase H and a 3'"'"' segment of nucleotides that do not activate RNase H, wherein the 5'"'"' segment of nucleotides comprises nucleotide phosphorothioates and the 3'"'"' segment of nucleotides comprises nucleotide alkylphosphonates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

12. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that activate RNase H and a 3'"'"' segment of nucleotides that do not activate RNase H, wherein the 5'"'"' segment of nucleotides comprises nucleotide phosphorothioates, and the 3'"'"' segment of nucleotides comprises nucleotide phosphoromorpholidates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the RNA segment.

13. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that activate RNase H and a 3'"'"' segment of nucleotides that do not activate RNase H, wherein the 5'"'"' segment of nucleotides comprises nucleotide phosphorothioates, and the 3'"'"' segment of nucleotides comprises nucleotide phosphoramidates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

14. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that activate RNase H and a 3'"'"' segment of of nucleotides that activate RNase H, wherein 5'"'"' segment of nucleotides comprises nucleotide alkylphosphonates, and the 3'"'"' segment of nucleotides comprises nucleotide phosphorothioates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

15. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that activate RNase H and a 3'"'"' segment of of nucleotides that activate RNase H, wherein the 5'"'"' segment of nucleotides comprises nucleotide phosphoromorpholidates, and the 3'"'"' segment of nucleotides comprises nucleotide phosphorothioates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby cleaving the target RNA segment.

16. A method of site-specific cleavage of a target RNA segment of an RNA molecule consisting essentially of combining the RNA molecule with a mixed phosphate backbone oligonucleotide that is complementary to all or a portion of the RNA molecule in the presence of RNase H, the mixed phosphate backbone oligonucleotide comprising a 5'"'"' segment of nucleotides that do not activate RNase H and a 3'"'"' segment of nucleotides that activate RNase H, wherein the 5'"'"' segment of nucleotides comprises nucleotide phosphoramidates, and the 3'"'"' segment of nucleotides comprises nucleotide phosphorothioates, wherein said combining is under conditions appropriate for hybridization of complementary nucleotide sequences and activation RNase H, thereby cleaving the target RNA segment.