Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-25
2001-10-23
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091310, C435S091100, C435S091510, C514S04400A
Reexamination Certificate
active
06306596
ABSTRACT:
BACKGROUND
In recent years, a number of methods have been developed for manipulating DNA. Some of these methods employ biomolecules to cut or cleave DNA, which in some instances renders the substrate DNA nonfunctional. Other methods employ biomolecules to facilitate insertion of new pieces of nucleic acid into the cleavage site of the DNA substrate. The insertion of new segments of nucleic acid into the cleavage sites of the DNA substrate changes the characteristics of the RNA or protein molecules encoded by the substrate DNA molecules. Accordingly, the biomolecules which catalyze the cleavage of DNA substrates or the insertion of new nucleic acid molecules into the DNA substrates are useful tools for genetic engineering, for analytical studies and for diagnostic studies. One such molecule used for cleaving DNA substrates is the restriction endonuclease.
Restriction endonucleases are enzymatic proteins that cleave double-stranded DNA. Such endonucleases recognize specific nucleotide sequences in double-stranded DNA, and cleave both strands within or near the specific recognition site. Such specificity renders the restriction endonucleases important tools in the controlled fragmentation of double-stranded DNA. Restriction endonucleases are also useful analytical tools for determining whether certain sequences are present in substrate DNA and in genomic sequencing studies.
However, restriction endonucleases only cleave DNA substrates; they do not insert new nucleic acid molecules into the cleaved DNA substrate. Accordingly, another biomolecule is needed to insert new pieces of DNA or RNA into the double-stranded DNA.
Ribozymes are catalytic RNA molecules that cleave RNA and, in certain circumstances, that insert new pieces of RNA into the cleavage site of the RNA substrate. Unfortunately, ribozymes have not been particularly useful for cleaving single-stranded DNA substrates or double-stranded DNA substrates. Ribozymes cut single-stranded DNA only under extreme conditions of elevated temperatures and high concentrations of magnesium. Ribozymes can be used to cleave double-stranded DNA only after the DNA is denatured and separated into two pieces of single-stranded DNA. Moreover, ribozymes have limited use in systems containing ribonucleases.
Accordingly, it would be desirable to have methods which employ a new tool that is capable of cleaving double-stranded DNA molecules, single-stranded DNA molecules, and single-stranded RNA molecules at specific sites. Methods which employ a new biomolecule capable of cleaving RNA molecules, single-stranded DNA molecules and double-stranded DNA molecules at specific sites and simultaneously inserting a new nucleic acid molecule into the cleavage site are especially desirable.
SUMMARY OF THE INVENTION
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 nucleic acid molecules into the cleaved substrate. The nucleotide integrase is a ribonucloeprotein particle comprising a group II intron RNA and a group II intron-encoded protein, which is bound to the group II intron RNA.
One method uses a nucleotide integrase to cleave one strand, hereinafter referred to as the “top 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, “EBS1” and “EBS2”, that are capable of hybridizing with two intron RNA binding sequences,“IBS1” and “IBS2”, respectively, 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 under conditions that 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. Preferably, the nucleotide immediately preceding the first nucleotide of the EBS1 sequence on the group II intron RNA, hereinafter referred to as the &dgr; nucleotide is complementary to the nucleotide at +1 on the top strand of the substrate, hereinafter referred to as the &dgr;′ nucleotide.
As denoted herein, nucleotides that are located upstream of the cleavage site have a (−) position relative to the cleavage site, and nucleotides that are located downstream of the cleavage site have a (+) position relative to the cleavage site. Thus, in the above-described method, the cleavage site is located between nucleotides −1 and +1 on the top strand of the double-stranded DNA substrate. The IBS1 sequence and the IBS2 sequence lie in a region of the recognition site which extends from about position −1 to about position −14 relative to the cleavage site. As denoted herein, the first sequence element comprises from about 10 to about 12 pairs of nucleotides that lie upstream of IBS2 and IBS1, i.e from about position −12 relative to the cleavage site to about position −26 relative to the cleavage site. As denoted herein, the second sequence element comprises from about 10 to about 12 pairs of nucleotides that lie downstream of the cleavage site, i.e., at positions +1 to about +12. The EBS1 sequence of the group II intron RNA comprises from about 5 to 7 nucleotides and has substantial complementarity with the nucleotides at positions −1 to about −5 or about −7 on the top strand of the DNA substrate. The EBS2 sequence comprises from about 4 to 7 nucleotides and has substantial complementarity with the nucleotides at positions from about −6 to about −14 on the top strand of the DNA substrate.
The present invention also provides a method which employs a nucleotide integrase to cleave both strands 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, EBS1 and EBS2, that are capable of hybridizing with two intron RNA binding sequences, IBS1 and IBS2, on the top 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 top strand. As denoted herein, the second sequence element comprises from about 10 to about 12 pairs of nucleotides that lie downstream of the cleavage site, i.e from position +1 to about position +10, +11, or +12. Preferably, the &dgr; nucleotide of the group II intron RNA is complementary to the &dgr;′ nucleotide on the top strand of the substrate.
Another method provided by the present invention employs a nucleotide integrase for cleaving a single stranded nucleic acid substrate and for inserting the group II intron RNA of the nucleotide integrase into the cleavage site. The method comprises the steps of: providing a nucleotide integrase having two hybridizing sequences, EBS1 and EBS2, that are capable of hybridizing with two intron RNA-binding sequences, IBS1 and IBS2, 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. The EBS1 sequence of the group II intron RNA comprises from about 5 to 7 nucleotides that have substantial complementarity with the nucleotides at positions −1 to about −5 or about −7 relative to the putative cleavage site. The EBS2 sequence comprises from about 4 to 7 nucleotides that have substantial complementarity with the nucleotides at positions from about &minus
Beall Clifford James
Guo Huatao
Lambowitz Allen M.
Mohr Georg
Zimmerly Steven
Arthur Lisa B.
Calfee Halter & Griswold LLP
Goldberg Jeanine
The Ohio State University Research Foundation
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