Nucleic acid enzyme for rna cleavage

Details Nucleic acid enzyme for rna cleavage Nucleic acid enzyme for rna cleavage

C - Chemistry, Metallurgy

12

N

C12N 15/11 (2006.01) C12N 9/00 (2006.01) A61K 38/00 (2006.01)

Patent

CA 2330570

A method is described for cleaving a nucleic acid substrate with a nucleic acid enzyme at a cleavage site comprising mixing the substrate with the enzyme, wherein the substrate includes a 7 nucleotide sequence with at least 6 nucleotides (3') to the cleavage site and at least I nucleotide (5') to the cleavage site and of formula: 5'-H'.dwnarw.GNNHNN-3' wherein each N is a nucleotide which may be the same or different, H is a nucleotide selected from the group consisting of A, U, C, and T, and j is the site of cleavage, and H' is a ribonucleotide selected from the group consisting of A, U, and C, wherein (i) the first nucleotide 3' to the cleavage site is capable of forming a wobble pair with the enzyme, (ii) the second, third, fifth, and sixth nucleotides 3' to the cleavage site are capable of forming conventional Watson-Crick base pairs with the enzyme, (iii) the fourth nucleotide 3' to the cleavage site is capable of forming a non-conventional Watson-Crick base pair with the enzyme, and (iv) the first nucleotide 5' to the cleavage site does not form a base pair with the enzyme; and the enzyme comprises a substrate binding portion which is capable of base pairing to the 6 nucleotides 3' to the cleavage site of the substrate and which binding portion comprises the sequence: 3'-UNNXNN-5' wherein each N is a nucleotide which may be the same or different, and X is a nucleotide selected from the group consisting of T, U, A, and G, whereby binding of the substrate to the enzyme effects cleavage of the substrate at the cleavage site.

L'invention porte sur un procédé de coupure d'un substrat d'acide nucléique par une enzyme d'acide nucléique en un site de coupure consistant à mélanger le substrat à l'enzyme. Le substrat comporte au niveau du site de coupure une séquence d'au moins 7 nucléotides dont au moins 6 nucléotides 3', et un nucléotide 5' de formule 5'-H' &darr& GNNHNN-3', dans laquelle: chaque N est un nucléotide identique ou différent; H est un nucléotide choisi parmi A, U, C et T; &darr& est le site de coupure; H' est un ribonucléotide choisi parmi A, U et C, et au sujet de laquelle: (i) le premier nucléotide 3' du site de coupure peut former une paire flottante avec l'enzyme, (ii) les deuxième, troisième, cinquième et sixième nucléotides 3' du site de coupure peuvent former avec l'enzyme des paires de base de Watson-Crick conventionnelles, (iii) le quatrième nucléotide 3' du site de coupure peut former avec l'enzyme des paires de base de Watson-Crick non conventionnelles, et (iv) le premier nucléotide 5' du site de coupure ne peut former de paire de base avec l'enzyme; de plus, l'enzyme comporte une portion se fixant au substrat et capable de former une paire de base avec les six nucléotide 3' du site de coupure du substrat, ladite portion comportant la séquence 3'-UNNXNN-5' dans laquelle chaque N est un nucléotide identique ou différent; X est un nucléotide choisi parmi T, U, A et G et qui lorsque le substrat s'y fixe entraîne la coupure du substrat au site de coupure.

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