Catalytic RNA molecules

• US 6,063,566 A
• Filed: 07/17/1996
• Issued: 05/16/2000
• Est. Priority Date: 05/13/1994
• Status: Expired due to Fees

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Joyce, Gerald F.
• Primary Examiner(s): LeGuyader, John L.
• Application Number:
  US08/682,423
• Time in Patent Office: 1,399 Days
• Field of Search: 435/6, 435/91.31, 435/172.1, 435/240.2, 435/240.1, 435/252.3, 536/23.1, 536/23.2, 536/24.5, 514/44
• US Class Current: 435/6.12
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/124 based on group I or II introns
• CPC Class Codes: C12N 2310/1241 Tetrahymena

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.

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_D) of 0.06 to 1 micromolar (μ

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→

A;

51/52-insert AGAA;

87;

A→

deleted;

94;

A→

U,

94;

A→

C;

115;

A→

U;

116;

G→

A;

138;

C→

A;

166;

C→

A;

167;

U→

G;

170;

C→

U;

188;

G→

A;

190;

U→

A;

191;

G→

U;

205;

U→

C;

215;

G→

A;

239;

U→

A;

258;

U→

C;

312;

G→

A;

313;

G→

U;

313;

G→

C;

31.4;

A→

G;

317;

U→

G;

317;

U→

C;

317;

U→

A;

333;

U→

C;

350;

C→

U; and

364;

C→

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_D) of 0.06 to 1 micromolar (μ

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→

A;

51/52;

CUAA insert;

86/87;

UA→

del;

94;

A→

U;

115/116;

AG→

UA;

165;

C→

U;

170;

C→

U;

188;

C→

A;

190/191;

UG→

AU, 205;

U→

C;

215;

G→

A;

239;

U→

A;

271;

U→

C;

289;

U→

A;

312;

G→

AA;

340U→

A;

364;

C→

U; and

366;

G→

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_D) of 0.06 to 1 micromolar (μ

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→

A;

51;

G→

A,

51/52;

CUAA insert;

86/87;

UA→

del;

94;

A→

U ;

115/116;

AG→

UA;

138;

C→

A;

165C→

U;

170;

C→

U;

175;

G→

A;

188;

G→

A;

190/191 UG→

AU,

205;

U→

C;

215;

G→

A;

239;

U→

A;

271;

U→

C;

288/289;

GU→

AA;

292;

U→

A;

312;

G→

AA;

334;

A→

G;

340;

U→

A;

347;

A→

G;

366;

G→

A;

389;

U→

C; and

390;

A→

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_D) of 0.06 to 1 micromolar (μ

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_D) of 0.06 to 1 micromolar (μ

M);

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

e. amplifying said selected separated individuals.