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Last Updated: March 29, 2024

Claims for Patent: 6,027,895


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Summary for Patent: 6,027,895
Title: Methods for cleaving DNA with nucleotide integrases
Abstract:The present invention provides new methods, employing a nucleotide integrase, for cleaving single-stranded RNA substrates, single-stranded DNA substrates, and double- stranded DNA substrates at specific sites and for inserting a nucleic acid molecule into the cleaved substrate. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences that are capable of hybridizing with two intron RNA binding sequences on the one strand of the DNA substrate, and a group II-intron encoded protein which binds to a first sequence element of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate under conditions that permit the nucleotide integrase to cleave the one strand of the DNA substrate and to insert the group II intron RNA into the cleavage site. The method of cleaving both strands of a double-stranded DNA substrate comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences that are capable of hybridizing with two intron RNA binding sequences on one strand of the substrate, and a group II-intron encoded protein that is capable of binding to a first sequence element and to a second sequence element in the recognition site of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate such that the nucleotide integrase cleaves both strands of the DNA substrate and inserts the group II intron RNA into the cleavage site of the one strand. The method for cleaving a single-stranded nucleic acid substrate comprises the steps of: providing a nucleotide integrase having two hybridizing sequences that are capable of hybridizing with two intron RNA-binding sequences on the single-stranded substrate, and a group II intron encoded protein; and reacting the nucleotide integrase with the single stranded nucleic acid substrate for a time and at a temperature sufficient to allow the nucleotide integrase to cleave the substrate and to attach the group II intron RNA molecule thereto.
Inventor(s): Lambowitz; Allen M. (Austin, TX), Zimmerly; Steven (Calgary, CA), Gau; Huatao (Austin, TX), Mohr; Georg (Austin, TX), Beall; Clifford James (Columbus, OH)
Assignee: The Ohio State University Research Foundation (Columbus, OH)
Application Number:09/031,897
Patent Claims:1. A method of cleaving a double stranded DNA substrate at a cleavage site, said substrate having a recognition site, said method comprising the following steps:

(a) providing a nucleotide integrase comprising;

(i) a group II intron RNA having a first hybridization sequence capable of hybridizing with a first intron RNA binding sequence of one strand of the DNA substrate and a second hybridization sequence capable of hybridizing with a second RNA binding sequence on said one strand of the substrate; and

(ii) a group II intron-encoded protein capable of binding with at least one nucleotide in a first sequence element in the recognition site of the substrate, said group II intron-encoded protein being bound to said group II intron RNA; and

(b) reacting the nucleotide integrase with the substrate to permit the nucleotide integrase to cleave said one strand of the DNA substrate and to insert the group II intron RNA into the cleavage site.

2. The method of claim 1 wherein there is at least 80% complementarity between the first hybridization sequence and the first intron RNA binding sequence and at least 80% complementarity between the second hybridization sequence and the second intron RNA-binding sequence.

3. The method of claim 1 wherein the group II intron RNA further comprises a .delta. nucleotide that is complementary to a .delta.' nucleotide on said one strand of the substrate, said .delta.' nucleotide being located at position +1 relative to the cleavage site.

4. The method of claim I wherein the group II intron RNA is a wild-type or modified aI2 intron RNA and wherein the group II intron-encoded protein is an aI2 intron-encoded protein.

5. The method of claim 4 wherein said one strand of the substrate comprises a T at position -13 relative to the cleavage site, a T at position -15 relative to the cleavage site, a C at position -18 relative to the cleavage site, and a G at position -16 or position -19 relative to the cleavage site.

6. The method of claim 4 wherein said one strand of said substrate comprises a G at -19, a C at -18, a G at -16, a T at -15, and a T at -13 relative to the cleavage site.

7. The method of claim 1 wherein the group II intron RNA is a wild-type or modified aI1 intron RNA and wherein said group II intron-encoded protein is a protein encoded by an aI1 intron.

8. The method of claim 7 wherein said one strand of the substrate has a C at -13 relative to the cleavage site.

9. The method of claim 7 wherein said one strand of the substrate comprises a G at -22, a G at -21, an A at -19, an A at -18, and a C at -13 relative to the cleavage site.

10. A method of cleaving a double stranded DNA substrate at a cleavage site, said substrate having a recognition site, said method comprising the following steps

(a) providing a nucleotide integrase comprising;

(i) a wild-type or modified Ll.ltrB intron RNA, wherein said intron RNA has a first hybridization sequence capable of hybridizing with a first intron RNA binding sequence of one strand of the DNA substrate and a second hybridization sequence capable of hybridizing with a second RNA binding sequence on said one strand of the substrate; and

(ii) a protein encoded by the Ll.ltrB intron, said protein capable of binding with at least one nucleotide in a first sequence element in the recognition site of the substrate, said protein being bound to said intron RNA; and

(b) reacting the nucleotide integrase with the substrate to permit the nucleotide integrase to cleave said one strand of the DNA substrate and to insert the intron RNA into the cleavage site.

11. The method of claim 10 wherein said one strand of the substrate comprises a G at -21 and an A at -20 relative to the cleavage site.

12. The method of claim 11 wherein said one strand of the substrate comprises a G at -21, an A at -20, a T at -19, a G at -17 and a G at -15 relative to the cleavage site.

13. A method of cleaving a single-stranded nucleic acid substrate at a cleavage site comprising the following steps:

(a) providing a nucleotide integrase comprising;

(i) a group II intron RNA having a first hybridizing sequence capable of hybridizing with a first intron RNA binding sequence on the nucleic acid substrate and a second hybridizing sequence capable of hybridizing with a second intron RNA binding sequence on said nucleic acid substrate, and

(ii) a group II intron-encoded protein bound to said group II intron RNA; and

(b) reacting the nucleotide integrase with the substrate to permit the nucleotide integrase to cleave the nucleic acid substrate and to insert the group II intron RNA into the cleavage site.

14. The method of claim 13 wherein the substrate is RNA.

15. The method of claim 13 wherein the substrate is DNA.

16. The method of claim 13 wherein the nucleotide integrase is selected from a group consisting of:

(a) a wild-type or modified aI2 intron RNA and an aI2 intron-encoded protein;

(b) a wild-type or modified aI1 intron RNA and an aI1 intron-encoded protein; and

(c) a wild-type or modified Ll.ltrB intron RNA and an Ll.ltrB intron-encoded protein.

17. A method of cleaving both strands of a double-stranded DNA substrate comprising the following steps:

(a) providing a nucleotide integrase comprising;

(i) a group II intron RNA having a first hybridizing sequence capable of hybridizing with a first intron RNA binding sequence on one strand of the DNA substrate and a second hybridizing sequence capable of hybridizing with a second intron RNA binding sequence on said one strand of the DNA substrate; and

(ii) a group II intron-encoded protein capable of binding to at least one nucleotide in a first sequence element and to at least one nucleotide in a second sequence element of the substrate, said group II intron-encoded protein being bound to said group II intron RNA; and

(c) reacting the nucleotide integrase with the substrate for a time and at a temperature sufficient to permit the nucleotide integrase to cleave both strands of the DNA substrate and to insert the group II intron RNA into the cleavage site on said one strand.

18. The method of claim 17 wherein the nucleotide integrase is selected from a group consisting of:

(a) a wild-type or modified aI2 intron RNA and an aI2 intron-encoded protein;

(b) a wild-type or modified aI1 intron RNA and an aI1 intron-encoded protein; and

(c) a wild-type or modified Lltr.B intron RNA and an Ll.ltrB intron-encoded protein.

19. The method of claim 17 wherein there is at least 80% complementarity between the first hybridization sequence and the first intron RNA binding sequence and wherein there is at least 80% complementarity between the second hybridization sequence and the second intron RNA binding sequence.

20. The method of claim 17 wherein the group II intron RNA is a wild-type or modified aI2 intron RNA, wherein the group II intron-encoded protein is an aI2 intron-encoded protein; and wherein said one strand of the substrate comprises a C at -18, a T at -15, a T at -13, a G at -13 or -16, a T at +1, a T at +4 and a G at +4.

21. The method of claim 17 wherein the group II intron RNA is a wild-type or modified aI1 intron RNA: wherein the group II intron-encoded protein is a protein encoded by an aI1 intron; and wherein said one strand of the substrate comprises a C at -13, a T at +1, a T at +2, a T at +3, a T at +4, an A at +5, a G at +6, a T at +7, and an A at +8.

22. A method of cleaving both strands of a double-stranded DNA substrate comprising the following steps:

(a) providing a nucleotide integrase comprising;

(i) a wild-type or modified Ll.ltrB intron RNA, said intron RNA having a first hybridizing sequence capable of hybridizing with a first intron RNA binding sequence on one strand of the DNA substrate and a second hybridizing sequence capable of hybridizing with a second intron RNA binding sequence on said one strand of the DNA substrate; and

(ii) a protein encoded by an Ll.ltrB intron, said protein capable of binding to at least one nucleotide in a first sequence element and to at least one nucleotide in a second sequence element of the substrate, said protein being bound to said intron RNA; and

(c) reacting the nucleotide integrase with the substrate for a time and at a temperature sufficient to permit the nucleotide integrase to cleave both strands of the DNA substrate and to insert the group II intron RNA into the cleavage site on said one strand.

23. The method of claim 17 wherein the group II intron encoded protein comprises a reverse transcriptase domain, and wherein the nucleotide integrase and the substrate are reacted in a reaction mixture comprising dATP, dGTP, dTTP, and dCTP such that a cDNA molecule is formed in the cleavage site on the other strand of the DNA substrate.

24. A method of detecting the presence of a nucleotide recognition site in a nucleic acid substrate comprising the steps of:

(a) providing a nucleotide integrase capable of cleaving a nucleic acid substrate having a recognition site;

(b) reacting the nucleic acid substrate with said nucleotide integrase; and

(c) assaying for cleavage of the nucleic acid substrate, wherein cleavage is indicative of the presence of the recognition site in the nucleic acid substrate.

25. The method of claim 10 wherein there is at least 80% complementarity between the first hybridization sequence and the first intron RNA binding sequence and at least 80% complementarity between the second hybridization sequence and the second intron RNA-binding sequence.

26. A method of cleaving a single-stranded nucleic acid substrate at a cleavage site comprising the following steps:

(a) providing a nucleotide integrase comprising;

(i) a wild-type or modified Lltr.B intron RNA, said intron RNA having a first hybridizing sequence capable of hybridizing with a first intron RNA binding sequence on the nucleic acid substrate and a second hybridizing sequence capable of hybridizing with a second intron RNA binding sequence on said nucleic acid substrate, and

(ii) an Ll.ltrB intron-encoded protein bound to said intron RNA; and

(b) reacting the nucleotide integrase with the substrate to permit the nucleotide integrase to cleave the nucleic acid substrate and to insert the intron RNA into the cleavage site.

27. The method of claim 22 wherein the top strand of the substrate comprises a G at -21, an A at -20, a C at +1, an A at +2, a T at +3, an A at +4, a T at +5, a C at +6, an A at +7, and a T at +8.

Details for Patent 6,027,895

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 06/04/1986 ⤷  Try a Trial 2015-09-12
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 ⤷  Try a Trial 2015-09-12
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b Injection 103132 ⤷  Try a Trial 2015-09-12
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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