Claims for Patent: 6,306,596

Summary for Patent: 6,306,596

Title:	Methods for cleaving single-stranded and double-stranded DNA substrates with nucleotide integrase
Abstract:	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 are provided. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing an isolated nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on the top 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 to permit the nucleotide integrase to cleave the top 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 for hybridizing with two intron RNA binding sequences on one strand of the substrate, and a group II-intron encoded protein that interacts with a first sequence element and 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 for 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 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), Guo; Huatao (Austin, TX), Mohr; Georg (Austin, TX), Beall; Clifford James (Columbus, OH)
Assignee:	The Ohio State University Research Foundation (Columbus, OH)
Application Number:	09/257,770
Patent Claims:	1. A method of cleaving a double stranded DNA substrate at a cleavage site comprising the following steps: (a) providing a double-stranded DNA substrate having a top strand and a recognition site; wherein said top strand has a T at position -4 relative to the cleavage site; (b) providing an isolated nucleotide integrase comprising (i) a wild-type or a modified Ll.ltrB intron RNA having a first hybridization sequence for hybridizing with a first intron RNA binding sequence on said top strand of the DNA substrate and a second hybridization sequence for hybridizing with a second RNA binding sequence on said top strand of the substrate; and (ii) a protein encoded by an Ll.ltrB intron for binding with at least one nucleotide in a first sequence element in the recognition site of the substrate, said protein being bound to said Ll.ltrB intron RNA; and (c) reacting the nucleotide integrase with the substrate to permit the nucleotide integrase to cleave said top strand of the DNA substrate and to insert the Ll.ltrB intron RNA into the cleavage site. 2. The method of claim 1 wherein the top strand of the substrate further has a G at -21 and an A at -20 relative to the cleavage site and wherein the top strand of the substrate has a sequence which differs from the wt sequence, SEQ. ID. NO: 4, shown in FIG. 8. 3. The method of claim 2 wherein the top strand of the substrate further has a T at -19, a G at -17, and a G at -15 relative to the cleavage site. 4. The method of claim 2 wherein the nucleotide at -3, or -5, or -7 on the top strand of the substrate is not complementary to a nucleotide at the corresponding position in the first hybridization sequence of the Ll.ltrB intron RNA. 5. The method of claim 1 wherein the sequence of nucleotides from +1 through +5 on the top strand of the substrate contains more A and T nucleotides than C and G nucleotides. 6. The method of claim 1 wherein the sequence of nucleotides from -14 to -26 on the top strand of the substrate contains more A and T nucleotides than C and G nucleotides. 7. The method of claim 1 wherein the top strand of the substrate further has a C at -12, an A at -11, a C at -10, a C at -6, and a C at +1 relative to the cleavage site and wherein the top strand of the substrate has a sequence which differs from the wt sequence, SEQ. ID. NO: 4, shown in FIG. 8. 8. The method of claim 1 wherein the top strand of the substrate has a nucleotide that is complementary to the nucleotide on said top strand of the substrate, said nucleotide being located at position +1 relative to the cleavage site and wherein the top strand of the substrate has a sequence which differs from the wt sequence, SEQ. ID. NO: 4, shown in FIG. 8. 9. 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 and wherein the top strand of the substrate has a sequence which differs from the wt sequence, SEQ. ID. NO: 4, shown in FIG. 8. 10. A method for cleaving a single-stranded nucleic acid substrate at a cleavage site comprising the following steps: (a) providing an isolated nucleotide integrase comprising: (i) a group II intron RNA having a first hybridizing sequence for hybridizing with a first intron RNA binding sequence on the nucleic acid substrate and a second hybridizing sequence for hybridizing with a second hybridizing sequence on said nucleic acid substrate, wherein the three nucleotides immediately upstream of the first hybridizing sequence are complementary to nucleotides at positions +1 to +3 on the 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.

Details for Patent 6,306,596

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		2018-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2018-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2018-02-26