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

• US 5,149,797 A
• Filed: 02/15/1990
• Issued: 09/22/1992
• 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: Agrawal, Sudhir, Pederson, Thoru, Zamecnik, Paul C., Mayrand, Sandra
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US07/480,269
• Time in Patent Office: 950 Days
• Field of Search: 435/172.3, 435/172.1, 435/69.1-69.9, 435/183, 536/27, 935/39-41
• US Class Current: 536/23.1
• 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 mixed phosphate backbone oligonucleotide consisting essentially of an internal segment of modified nucleotides which activates RNase H and two modified nucleotide sequences which do not activate RNAse H, in which the two modified nucleotide sequences flank the internal segments, one on each side of the internal segment, wherein the internucleoside bridging phosphate residues of the internal segment are modified phosphates which are phosphorothioates and the internucleoside bridging phosphate residues of the two flanking modified nucleotide sequences are modified phosphate selected from the group consisting of:

methyl phosphonates, phosphoromorpholidates, phosphoropiperazidates, and phosphoramidates.

2. The mixed phosphate backbone oligonucleotide of claim 1, wherein the internucleoside bridging phosphate residues of the two flanking modified nucleotide sequences are methyl phosphonates.

3. The mixed phosphate backbone oligonucleotide of claim 1, wherein the internucleoside bridging phosphate residues of the two flanking modified nucleotide sequences are phosphoromorpholidates.

4. The mixed phosphate backbone oligonucleotide of claim 1, wherein the internucleoside bridging phosphate residues of the two flanking modified nucleotide sequences are phosphoropiperazidates.

5. The mixed phosphate backbone oligonucleotide of claim 1, wherein the internucleoside bridging phosphate residues of the two flanking modified nucleotide sequences are phosphoramidates.