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Generated: August 19, 2019

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Claims for Patent: 6,063,566

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Summary for Patent: 6,063,566
Title: Catalytic RNA molecules
Abstract:The present invention discloses nucleic acid enzymes capable of cleaving nucleic acid molecules, including single-stranded DNA, in a site-specific manner under physiologic conditions, as well as compositions including same. The present invention also discloses methods of making and using the disclosed enzymes and compositions. The present invention further discloses nucleic acid enzymes or catalytic (enzymatic) RNA molecules that are capable of cleaving a variety of bonds, including phosphodiester bonds and amide bonds, in a variety of substrates. Thus, various disclosed enzymatic RNA molecules are capable of functioning as nucleases, amidases, and/or peptidases. The present invention also relates to compositions containing the disclosed catalytic RNA molecules and to methods of making, selecting, and using such enzymes and compositions.
Inventor(s): Joyce; Gerald F. (Encinitas, CA)
Assignee: The Scripps Research Institute (CA)
Application Number:08/682,423
Patent Claims:1. A catalytic RNA molecule comprising a ribonucleotide polymer derived from a group I intron having the nucleotide sequence of SEQ ID NO 1, said polymer having a substrate binding affinity expressed as an equilibrium constant (K.sub.D) of 0.06 to 1 micromolar (.mu.M), wherein said polymer catalyzes the site-specific cleavage of substrate single-stranded deoxyribonucleic acid molecules under physiological conditions, wherein said polymer has one or more point mutations in its nucleotide sequence which improve one or more catalytic parameters of said catalytic RNA molecule over said group I intron, said mutation selected from the group consisting of:

44:G.fwdarw.A; 51/52-insert AGAA;

87:A.fwdarw.deleted; 94:A.fwdarw.U,

94:A.fwdarw.C; 115:A.fwdarw.U;

116:G.fwdarw.A; 138:C.fwdarw.A;

166:C.fwdarw.A; 167:U.fwdarw.G;

170;C.fwdarw.U; 188:G.fwdarw.A;

190:U.fwdarw.A; 191:G.fwdarw.U;

205:U.fwdarw.C; 215:G.fwdarw.A;

239:U.fwdarw.A; 258:U.fwdarw.C;

312:G.fwdarw.A; 313:G.fwdarw.U;

313:G.fwdarw.C; 31.4:A.fwdarw.G;

317:U.fwdarw.G; 317:U.fwdarw.C;

317:U.fwdarw.A; 333:U.fwdarw.C;

350:C.fwdarw.U; and

364:C.fwdarw.U.

2. A catalytic RNA molecule comprising a ribonucleotide polymer derived from a group I intron having the nucleotide sequence of SEQ ID NO 1, said polymer having a substrate binding affinity expressed as an equilibrium constant (K.sub.D) of 0.06 to 1 micromolar (.mu.M), wherein said polymer catalyzes the site-specific cleavage of substrate single-stranded deoxyribonucleic acid molecules under physiological conditions, wherein said polymer has one or more point mutations in its nucleotide sequence which improve one or more catalytic parameters of said catalytic RNA molecule over said group I intron, said mutation selected from the group consisting of:

44:G.fwdarw.A; 51/52:CUAA insert;

86/87: UA.fwdarw.del; 94:A.fwdarw.U;

115/116:AG.fwdarw.UA; 165:C.fwdarw.U;

170:C.fwdarw.U; 188:C.fwdarw.A;

190/191:UG.fwdarw.AU, 205:U.fwdarw.C;

215:G.fwdarw.A; 239:U.fwdarw.A;

271:U.fwdarw.C; 289:U.fwdarw.A;

312:G.fwdarw.AA; 340U.fwdarw.A;

364:C.fwdarw.U; and 366:G.fwdarw.A.

3. A catalytic RNA molecule comprising a ribonucleotide polymer derived from a group I intron having the nucleotide sequence of SEQ ID NO 1, said polymer having a substrate binding affinity expressed as an equilibrium constant (K.sub.D) of 0.06 to 1 micromolar (.mu.M), wherein said polymer catalyzes the site-specific cleavage of substrate single-stranded deoxyribonucleic acid molecules under physiological conditions, wherein said polymer has one or more point mutations in its nucleotide sequence which improve one or more catalytic parameters of said catalytic RNA molecule over said group I intron, said mutation selected from the group consisting of:

44:G.fwdarw.A; 51:G.fwdarw.A,

51/52:CUAA insert; 86/87: UA.fwdarw.del;

94:A.fwdarw.U ; 115/116:AG.fwdarw.UA;

138:C.fwdarw.A; 165C.fwdarw.U;

170:C.fwdarw.U; 175:G.fwdarw.A;

188:G.fwdarw.A; 190/191 UG.fwdarw.AU,

205:U.fwdarw.C; 215:G.fwdarw.A;

239:U.fwdarw.A; 271:U.fwdarw.C;

288/289:GU.fwdarw.AA; 292:U.fwdarw.A;

312:G.fwdarw.AA; 334:A.fwdarw.G;

340:U.fwdarw.A; 347:A.fwdarw.G;

366:G.fwdarw.A; 389:U.fwdarw.C; and

390:A.fwdarw.C.

4. A catalytic RNA molecule comprising a ribonucleotide polymer derived from a group I intron having the nucleotide sequence of SEQ ID NO 1, said polymer having a substrate binding affinity expressed as an equilibrium constant (K.sub.D) of 0.06 to 1 micromolar (.mu.M), wherein said polymer catalyzes the site-specific cleavage of substrate single-stranded deoxyribonucleic acid molecules under physiological conditions, and wherein said polymer sequence has the substitution of C for U at nucleotide position 271 and the substitution or AA for G at position 312.

5. A method of engineering catalytic RNA molecules in vitro that specifically cleave single-stranded DNA under physiological conditions, comprising the following steps:

a. obtaining a population of group I introns;

b. introducing genetic variation into said population to produce a variant population;

c. selecting individuals from said variant population that meet predetermined selection criteria including a deoxyribonucleic acid substrate binding affinity expressed as an equilibrium constant (K.sub.D)

of 0.06 to 1 micromolar (.mu.M);

d. separating said selected individuals from the remainder of said variant population; and

e. amplifying said selected separated individuals.

Details for Patent 6,063,566

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Schering INTRON A interferon alfa-2b VIAL 103132 001 1986-06-04   Try a Free Trial The Scripps Research Institute (CA) 2014-05-13 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 002 1986-06-04   Try a Free Trial The Scripps Research Institute (CA) 2014-05-13 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 003 1986-06-04   Try a Free Trial The Scripps Research Institute (CA) 2014-05-13 RX search
Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source

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