Enzymatic DNA molecules
AssigneeThe Scripps Research Institute
InventorsGerald F. Joyce, Ronald R. Breaker
Life of this patent
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Abstract
The present invention discloses deoxyribonucleic acid enzymes—catalytic or enzymatic DNA molecules—capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.
Claims68
A method of selecting a catalytic DNA molecule that cleaves a substrate nucleic acid sequence at a specific site, comprising the following steps:
- a. obtaining a population of single-stranded DNA molecules;
- b. admixing nucleotide-containing substrate molecules with said population of single-stranded DNA molecules to form an admixture;
- c. maintaining said admixture for a sufficient period of time and under predetermined reaction conditions to allow single-stranded DNA molecules in said population to cause cleavage of said substrate sequences, thereby producing substrate cleavage products;
- d. separating said population of single-stranded DNA molecules from said substrate sequences and substrate cleavage products;
- and
- e. isolating single-stranded DNA molecules that cleave nucleotide-containing substrate at a specific site from said population.
A method of in vitro selection of catalytic DNA molecules that cleave phosphoester bonds in a nucleic acid substrate, comprising the following steps:
- a. obtaining a population of single-stranded DNA molecules;
- b. introducing genetic variation into said population to produce a variant population;
- c. selecting individuals from said variant population that meet predetermined selection criteria;
- d. separating said selected individuals from the remainder of said variant population;
- and
- e. amplifying said selected individuals, thereby obtaining in vitro selected catalytic DNA molecules that cleave phosphoester bonds in a nucleic acid substrate.
A non-naturally-occurring catalytic DNA molecule comprising a nucleotide sequence defining a conserved core flanked by one or more recognition domains, variable regions, and spacer regions.
A catalytic DNA molecule having site-specific endonuclease activity, wherein said molecule includes one or more hairpin loop structures.
A catalytic DNA molecule having site-specific endonuclease activity, wherein said substrate nucleic acid sequence is attached to said catalytic DNA molecule.
A catalytic DNA molecule having site-specific endonuclease activity, wherein said catalytic DNA molecule comprises a nucleotide sequence selected from the group consisting of:
- SEQ ID NOS 52 through 101.
A catalytic DNA molecule having site-specific endonuclease activity, wherein said catalytic DNA molecule has a substrate binding affinity of about 1 μM or less.
A catalytic DNA molecule having site-specific endonuclease activity, wherein said catalytic DNA molecule binds substrate with a KD of less than about 0.1 μM.
A catalytic DNA molecule having site-specific endonuclease activity, wherein said endonuclease activity is enhanced by the presence of a divalent cation.
A catalytic DNA molecule having site-specific endonuclease activity, wherein said endonuclease activity is enhanced by the presence of a monovalent cation.
A catalytic DNA molecule having site-specific endonuclease activity, wherein said catalytic DNA molecule comprises a conserved core flanked by first and second substrate binding regions and wherein one or more spacer nucleotides are present between said conserved core and said substrate binding region.
A composition comprising two or more populations of catalytic DNA molecules having site-specific endonuclease activity, wherein each population of catalytic DNA molecules cleaves a different nucleotide sequence in a substrate.
A composition comprising two or more populations of catalytic DNA molecules having site-specific endonuclease activity, wherein each population of catalytic DNA molecules recognizes a different substrate.
A method of cleaving a phosphoester bond, comprising:
- a. admixing a catalytic DNA molecule capable of cleaving a substrate nucleic acid sequence at a defined cleavage site with a phosphoester bond-containing nucleic acid substrate, to form a reaction admixture;
- b. maintaining said admixture under predetermined reaction conditions to allow said catalytic DNA molecule to cleave said phosphoester bond, thereby producing a population of nucleic acid substrate products;
- c. separating said products from said catalytic DNA molecule;
- and
- d. adding additional substrate to said catalytic DNA molecule to form a new reaction admixture.
A method of cleaving a phosphoester bond, comprising:
- a. admixing a catalytic DNA molecule capable of cleaving a substrate nucleic acid sequence at a defined cleavage site with a phosphoester bond-containing nucleic acid substrate, to form a reaction admixture;
- and
- b. maintaining said admixture under predetermined reaction conditions to allow said catalytic DNA molecule to cleave said phosphoester bond, thereby producing a population of nucleic acid substrate products, wherein said predetermined reaction conditions include the presence of a monovalent cation, a divalent cation, or both.
Dependencies are inferred from the claim text.