JP2002515759A - DNA cleavage method using nucleotide integrase - Google Patents

Abstract

  (57) [Summary] The present invention provides methods for cleaving a nucleic acid substrate at a specific site using a nucleotide integrase and inserting a nucleic acid molecule into the cleaved substrate. Methods for cleaving a single strand of a double-stranded DNA substrate include two hybridizing sequences capable of hybridizing to two intron RNA binding sequences on one strand of a Group II-intron RNA substrate) and Group II -Providing a nucleotide integrase comprising an intron-encoded protein (which binds to a first sequence element of the substrate). Methods for cleaving both strands of a double stranded DNA substrate include group II-intron RNAs (having two hybridizing sequences capable of hybridizing to two intron RNA binding sequences on one strand of the substrate) and group II. Providing a nucleotide integrase comprising a II-intron encoded protein (which can bind to the first and second sequence elements at the recognition site of the substrate). The method of cleaving a single-stranded nucleic acid substrate comprises providing an integrase having two hybridizing sequences capable of hybridizing to two intron RNA binding sequences of the substrate and a Group II intron-encoding protein.

Description

DETAILED DESCRIPTION OF THE INVENTION             DNA cleavage method using nucleotide integrase                                   background   In recent years, a number of methods have been developed for manipulating DNA. Of these methods Some use biomolecules that cut or cleave DNA, Renders the substrate DNA non-functional in some cases. Another approach is to use new nucleic acids. Biomolecules are used to facilitate insertion of such fragments into the cleavage site of the DNA substrate. Insertion of a new segment of nucleic acid into the cleavage site of a DNA substrate is controlled by the substrate DNA molecule. Changes the characteristics of the RNA or protein molecule being loaded. Therefore, cleavage of the DNA substrate Biomolecules that catalyze the disruption or insertion of new nucleic acid molecules into DNA substrates A useful tool for analytical research and diagnostic research. DNA group One such molecule used for quality cleavage is a restriction endonuclease. It is.   Restriction endonucleases are enzymatic proteins that cut double-stranded DNA. Such an endonuclease is capable of converting a specific nucleotide sequence in double-stranded DNA. Recognizes and cleaves both strands within or near this specific recognition site. Such specificity is attributed to the restriction endonuclease to control the double-stranded DNA It is an important tool in mentoring. Restriction endonucleases are also identified To determine if the sequence is present in the substrate DNA, and genomic sequencing It is a useful analytical tool in routine research.   However, restriction endonucleases only cleave DNA substrates; Do not insert new nucleic acid molecules into the cleaved DNA substrate. Therefore, another biomolecule is D Necessary for inserting a new fragment of NA or RNA into double-stranded DNA.   Ribozymes cleave RNA and, in certain circumstances, generate new fragments of RNA. A catalytic RNA molecule that is inserted into the cleavage site of an RNA substrate. Unfortunately, ribozymes Was not particularly useful for cleavage of single-stranded or double-stranded DNA substrates. Ribozymes reduce single-stranded DNA only at elevated temperatures and high concentrations of magnesium. Under extreme conditions only. Ribozymes are denatured DNA, and single-stranded DNA Can be used to cut double-stranded DNA only after being separated into two fragments of . In addition, ribozymes have been of limited use in systems containing ribonucleases.   Therefore, double-stranded DNA molecules, single-stranded DNA molecules, and single-stranded RNA It would be desirable to have a new way to use new tools that could sever the child. RN A, single-stranded, and double-stranded DNA molecules can be cut at specific sites and Methods using new biomolecules that can sometimes insert new nucleic acid molecules into their cleavage sites Is particularly desirable.                                Summary of the Invention   The present invention uses a single strand at a specific site using a nucleotide integrase. For cleaving RNA, single- and double-stranded DNA substrates, and for nuclear A new method is provided for inserting an acid molecule into a cleaved substrate. Nucleoti Integrase is a group II intron RNA and a group II intron RNA. Ribonucleoprotein particles that contain It is bound to II intron RNA.   One method is to cut one strand using nucleotide integrase and Hereinafter, this is referred to as the “top strand” of the double-stranded DNA substrate. Honcho As indicated in the text, the nucleotides located upstream of the top strand cleavage site are: A nucleotide having a position (-) corresponding to the cleavage site and located downstream of the cleavage site Reotide has a (+) position corresponding to the cleavage site. Therefore, there are two cleavage sites. Located between -1 and +1 nucleotides of the top strand of the single-stranded DNA substrate. Base The quality top strand comprises a first intron RNA binding sequence, hereafter referred to as Is referred to as the “IBS1” sequence, and the second intron RNA binding sequence is referred to hereinafter as the “IBS1” sequence. It is referred to below as the “IBS2” sequence. The IBS1 and IBS2 sequences are about -1 site relative to the cleavage site. Located in the region ranging from to about -14 sites. Located upstream of IBS2 and IBS1 Cleavage from the first 10-12 pairs of nucleotides, i.e. about -12 from the cleavage site Up to about position -24 relative to the site, hereinafter collectively referred to as `` the first sequence element ". The first 10-12 pairs of nucleotides located downstream of the cleavage site Hereinafter, it is collectively referred to as "second array element" in the specification.   The method of the invention comprises the following steps: The two hybridizing sequences "EBS1 "And" EBS2 "(these are the IBS1 sequence and IBS2 Nucleic acid comprising a Group II intron RNA having Otide integrase, and at least one of the first sequence elements of the substrate Providing a Group II intron-encoded protein that binds to a nucleotide A nucleotide integrase, a nucleotide integrase being DNA Cleave the top strand of the substrate and insert a Group II intron RNA at the cleavage site Reacting with a double-stranded DNA substrate under conditions that permit the reaction. Preferably A Group II intron RNA, hereinafter referred to as δ nucleotides The nucleotide immediately preceding the first nucleotide of the EBS1 sequence of Complementary to the +1 nucleotide of the strand, hereinbelow referred to as the δ 'nucleotide Referred to. The EBS1 sequence of Group II intron RNA is approximately 5-7 nucleobases Nucleosides, and at positions -1 to about -5 or about -7 of the top strand of the DNA substrate It has substantial complementarity with tide. The EBS2 sequence contains about 4-7 nucleotides, and Has substantial complementarity with nucleotides at about position -6 to about -14 of the top strand of the DNA substrate .   The present invention also provides for the incorporation of nucleotide integrase into both strands of a double-stranded DNA substrate. Provide a method to use for The method comprises the following steps: Group II intron RNA with hybridizing sequences EBS1 and EBS2 Containing nucleotide integrases (these are the two introductories on the top strand of the substrate) That can hybridize with the RNA binding sequences IBS1 and IBS2), At least one nucleotide of the sequence element of Group II a capable of binding to at least one nucleotide of the two sequence elements Providing an intron-encoded protein; and a nucleotide integrator The nucleotide integrase cleaves both strands of the DNA substrate, And a double-stranded DNA substrate so as to insert the Group II intron RNA into the top strand cleavage site. The step of reacting. Preferably, Group II is that the delta nucleotide of the intron RNA is , Complementary to the δ 'nucleotide on the top strand of the substrate.   Another method provided by the present invention is a method for converting nucleotide integrase to single-stranded For cleavage of nucleic acid substrates and for nucleotide integrase group II in Used to insert Tron RNA at the cleavage site. The method comprises the following steps: Nucleotide integrants with two hybridizing sequences, EBS1 and EBS2 Lase (these are two intron RNA binding sequences on a single-stranded substrate, IBS1 and And group II intron-encoded proteins Providing a nucleotide integrase with a single-stranded nucleic acid substrate; Nucleotide integrase cleaves the substrate, and group II intron RNA React for a time and temperature sufficient to allow the molecule to bind to it Process. The EBS1 sequence of the Group II intron RNA is -1 to about -5 relative to the putative cleavage site. About 5 to 7 nucleotides having substantial complementarity with the nucleotide at or about position -7. Including. The EBS2 sequence is substantially equivalent to nucleotides from about position -6 to about position -14 relative to the putative cleavage site. About 4-7 nucleotides with good complementarity. Preferably, group II int The δ nucleotide of the long RNA is complementary to the δ ′ nucleotide on the top strand of the substrate. It is a target.   The invention also relates to a method for determining whether a nucleic acid contains a particular recognition site. This method uses a nucleotide integrator capable of cleaving a nucleic acid containing a specific recognition site. Providing the enzyme; reacting the nucleotide integrase with the nucleic acid; And a step of assaying for nucleic acid cleavage (where nucleic acid cleavage involves recognition of the recognition site by the nucleic acid) To include).                             BRIEF DESCRIPTION OF THE FIGURES   FIG. the second intron of the cerevisiae mitochondrial C0X1 gene EBS sequence of loop II intron RNA (hereinafter referred to as “aI2 intron” RNA FIG. 3 is a diagram showing the interaction between the IBS sequence and the IBS sequence of a DNA substrate. Substrate The cleavage site is indicated by an arrow.   FIG. 2 shows the nucleotide sequence of the aI2 intron RNA (SEQ ID NO: 1) cerev Group II intron RNA of the first intron of the isiae mitochondrial C0X1 gene (Hereinafter referred to as “al1 intron” RNA (SEQ ID NO: 2) FIG. The label on the sequence is wild-type al1 intron RNA And the location of the EBS1 and EBS2 sequences of the wild-type aI2 intron RNA.   FIG. 3 shows cleavage by nucleotide integrase containing wild-type aI2 intron RNA. Sequence of the DNA substrate to be cleaved and the protein encoded thereby, 5 is a chart depicting the positions of point mutations made in the sequence of FIG.   FIG. 4 shows nucleotide integrase containing wild-type aI2 intron RNA and its Group having a mutation in the first sequence element by the protein encoded thereby 4 is a graph showing the relative degree of quality cut.   FIG. 5 shows nucleotide integrase containing wild-type aI2 intron RNA and aI2. 2 In the second sequence element by the protein encoded by the intron RNA 4 is a graph showing the relative degree of cleavage of a substrate having a mutation.   FIG. 6 shows cleavage by nucleotide integrase containing wild-type al1 intron RNA. Sequence of the DNA substrate to be cleaved and the protein encoded by the al1 intron RNA 5 is a chart depicting quality and the location of mutations made in this sequence.   FIG. 7 shows the nucleotide integrase containing wild-type al1 intron RNA and the nucleotide integrase. Cleavage of a substrate having a mutation upstream of the cleavage site by the encoded protein 6 is a graph showing the relative degree of disconnection.   FIG. 8 shows wild-type group II intron RNA (Lactococcus lactis ItrB gene). Hereinafter referred to as “LI.ItrB intron RNA”). The sequence of the DNA substrate cleaved by teglase, and the 2 is a chart depicting proteins (hereinafter, referred to as ltrA protein). .   FIG. 9 shows nucleotide integrase containing wild-type LI.ItrB intron RNA and Relative cleavage of substrates with mutations in the first sequence element by the ltrA and ltrA proteins It is a graph which shows a degree.   FIG. 10 shows the LI.ItrB intron DNA sequence and adjacent exons ltrBE1 and ltrBE2. Part of the nucleotide sequence (SEQ ID NO: 5) of the LI.ItrB intron Nucleotide sequence of the coding frame (SEQ ID NO: 6) and the ltrA protein Shows the amino acid sequence (SEQ ID NO: 7).DETAILED DESCRIPTION OF THE INVENTION   The present invention provides a nucleotide integrase for manipulating DNA and RNA substrates. Provide a new way to use.   One method uses a nucleotide integrase to double-strand at a specific site. Cuts one strand of DNA (hereinafter referred to as the top strand), and simultaneously cuts at the cleavage site A nucleic acid molecule including RNA is bound to the thus obtained strand. The DNA substrate is the top strand of the substrate and A first intron RNA binding sequence (IBS1) located upstream of the cleavage site, and D Top strand of NA substrate and second intron RNA binding site located upstream of IBS1 sequence It has a recognition site, including the column (IBS2). The recognition site is also located upstream of the IBS2 sequence A first sequence element. The first sequence element comprises about 10-12 pairs of nucleic acids. Including otide.   The method for cleaving the top strand of a double-stranded DNA substrate includes the following steps: the top strand of the DNA substrate EBS1 and IBS2 sequences that can hybridize with the IBS1 and IBS2 sequences, respectively. Integrators Including Group II Intron RNAs Having EBS2 and EBS2 Sequences And binds to at least one nucleotide in the first sequence element Providing a Group II intron-encoded protein; Nucleotide integrase cleaves the top strand of the DNA substrate and Sufficient time and temperature to allow the intron RNA to be inserted into the cleavage site Reacting the nucleotide integrase with the double-stranded DNA substrate. Preferably Indicates that the group II intron-encoded protein has more than one in the first sequence element. Binds to a nucleotide.   The nucleotide integrase used in this method is a excised Group II Includes Group II intron-encoded proteins that bind to intron RNA. glue The EBS1 and EBS2 sequences of the intron II intron RNA are at least 80%, preferably 90%, %, More preferably the complete homology between the IBS1 sequence and the IBS in the top strand of the substrate. It has for each of 2 sequences. Group II intron-encoded proteins are RT Includes non-conserved portions of the domain, X domain, and Zn domain.   EBS1 is located in domain 1 of the group II intron RNA and of the substrate IBS1 It contains about 5-7 nucleotides that can hybridize to the nucleotide. EBS2 is EBS1 Located in domain 1 of the Group II intron RNA upstream of It contains about 4-7 nucleotides that can hybridize to a nucleotide. Group II Inn The nucleotides in the EBS1 and EBS2 sequences of the TRON RNA are replaced by nucleotides in the IBS1 or IBS2 sequence. If each is not at least 80% homologous to the leotide, then the glue Intron RNA is modified to increase homology between EBS and IBS sequences. Changed. As shown in FIG. 1, the IBS1 sequence of the substrate is upstream of the cleavage site, and The IBS2 sequence of the substrate is upstream of the IBS1 sequence.   To effectively cleave the substrate, the first nucleus of EBS1 of the group II intron RNA The nucleotide immediately adjacent to the nucleotide, δ, is +1 to the nucleotide at the top strand Preferably they are complementary. Thus, the δ nucleotide is in the top strand of the substrate. If not complementary to the +1 nucleotide, the +1 nucleotide in the top strand of the substrate Group II intron RNA modified to include delta nucleotides complementary to otide Is done. The nucleotide integrase then reacts with the substrate. Use in this method Suitable nucleotide integrases for use include, for example, aI2 nucleotide integrase. Lase, al1 nucleotide integrase, and ltrA nucleotide integra Including   aI2 integrase is a wild-type or modified aI2 intron-encoded protein. S. bound to quality of the second intron of the cerevisiae mitochondrial C0X1 gene, Wild-type or modified group II intron RNA (hereinafter referred to as “aI2 int Ron "RNA). The sequence of the wild-type aI2 intron RNA is shown in FIG. Shown in number 1. Sequence of the protein encoded by wild-type aI2 intron RNA Is shown in SEQ ID NO: 3. The EBS1 of the aI2 intron RNA contains 6 nucleotides and And located at positions 2985 to 2990 of the sequence shown in SEQ ID NO: 1. Of wild-type aI2 intron RNA EBS1 has the sequence 5'-AGAAGA. The EBS2 sequence of the aI2 intron RNA is 6 nucleotides. And is located at positions 2935-2940. The EBS2 sequence of the wild-type aI2 intron RNA is Has the sequence 5′-UCAUUA.   aI2 nucleotide integrase binds to a putative cleavage site on its top strand. T at relative positions -15 and -13, C at position -18 relative to the putative cleavage site, And a substrate having a G at position -16 or -19 relative to the putative cleavage site. Used to cut. Therefore, the use of aI2 nucleotide integrase First, the sequence of the top strand of the substrate is examined, and the target sequence 5′GCXXTXT or Position the target sequence 5'XCXGTXT. Where X represents A, C, G or T, and Where A represents a nucleotide having an adenine base and G has a guanine base C indicates a nucleotide having a cytosine base, and T Indicates a nucleotide having a thymine base. Next, the EBS2 distribution of the aI2 intron RNA was The sequence is substantially downstream from the IBS2 sequence, i.e., one of these target sequences If not homologous to the sequence of the 6 nucleotides located, and / or The EBS1 sequence of the Ron RNA is substantially downstream of the IBS1 sequence, i.e., the IBS2 sequence. If it is not homologous to the 6 nucleotide sequence located at As such, EBS1 and EBS2 are modified to have substantial complementarity. Substrate Top chain is A at position -21, G at position -19, C at position -18, G at position -16, T at position -15, and T at position -13 , The efficiency of cleavage by aI2 nucleotide integrase is increased.   al1 nucleotide integrase may be removed, wild-type, or modified and removed. Left, S. The glue of the first intron of the cerevisiae mitochondrial C0X1 gene II intron RNA (hereinafter referred to as "aI1 intron" RNA) and , Wild-type or modified al1 intron-encoded proteins. al1 intro The sequence of the RNA is shown in FIG. 2 and SEQ ID NO: 2. code by al1 intron RNA The sequence of the protein to be loaded is shown in SEQ ID NO: 4. EBS1 sequence of al1 intron RNA Contains 6 nucleotides and is located at positions 426-431. Wild type al1 intron RN EBS1 of A has the sequence 5'-CGUUGA. The EBS2 sequence of the al1 intron RNA is 6 nucleotides Contains tide and is located at positions 376-381. EBS2 of wild-type al1 intron RNA is It has the sequence 5'-ACAAUU.   al1 nucleotide integrase binds to a putative cleavage site on its top strand Used to cleave the top strand of a double-stranded DNA substrate with a C at position -13 Is done. Preferably, the top strand of the substrate is at position -13 relative to the putative cleavage site. At position C, G at position -22, G at position -21, A at position -19 and A at position -18. al1 intron The EBS2 sequence of the RNA is downstream immediately adjacent to the IBS2 sequence, i.e., the -13 C nucleotide. If there is no substantial complementarity with the six nucleotide sequence present, and / or The ESBI sequence of the al1 intron has the IBS1 sequence, i.e., the downstream region adjacent to the IBS2 sequence. Substantial complementarity with a 6-nucleotide sequence upstream adjacent to and cleavage site If not, the EBS1 and EBS2 sequences of the group II intron RNA are As described, they are modified to have substantial complementarity.   ItrA nucleotide integrase is removed, wild-type, or Lactococcus lactis ltrB gene group Ll.ltrB group II Introduced below, referred to as "Ll.ltrB intron" RNA, and Alternatively, the modified Ll.ltrB intron-encoded protein is Call it quality. The sequence of the L1.ltrB intron RNA is shown in FIG. lt The sequence of the rA protein is shown in SEQ ID NO: 7. The EBS1 sequence of Ll.ltrB intron RNA is It contains 7 nucleotides and is located at positions 457-463. Wild-type Ll.ltrB intron The EBS1 sequence of the RNA has the sequence 5′-GUUGUGG. EBS2 of Ll.ltyB intron RNA is 6 Nucleotides and is located at positions 401-406, including position 406. Wild type Ll.ltrB The EBS2 sequence of the intron RNA has the sequence 5'AUGUGU. Top strand corresponds to cleavage site Have a G at position -21 and an A at position -20, the ltrA nucleotide Glucase is used to cleave the top strand of a double-stranded DNA substrate. ltrA Nucleus Otide integrase is G at position -21, A at position -20, T at position -19, G and -15 at position -17. With a G at the position, the top strand is more effectively cleaved.   Another method is to break both strands of double-stranded DNA, and To attach a Group II intron RNA molecule to the strand break, Use integrase. Nucleotide integrase is Includes Group II intron-encoded proteins bound to Group II intron RNA Where the group II intron RNA contains the IBS1 sequence on the top strand of the substrate and IBS It has an EBS1 sequence and an EBS2 sequence that are each substantially homologous to the two sequences. EBS 1 distribution The row contains about 5-7 nucleotides. The EBS2 sequence contains about 4-7 nucleotides. glue The EBS1 and EBS2 nucleotides of the intron II Non-homologous nucleotides are at least 80% non-homologous to leotide. Preferably, it is modified by recombinant technology. Preferably, group II int Ron's delta nucleotide is homologous to the nucleotide at position +1 of the top strand. δ nu K If the leotide is not homologous to the nucleotide at position +1 then preferably the delta nucleotide Are modified homologously. Group II intron coat protein is the RT domain , X, and Zn domains. Substrate To insert cDNA at the cleavage site on the Tom strand, a Group II intron-coded The protein also contains a reverse transcription domain.   A method for cleaving both strands of a double-stranded DNA sequence having a recognition site includes the following steps. MM: High in the two intron RNA binding sequences of the top strand of the DNA substrate, IBS1 and IBS2. Group II intron RN with two hybridizable sequences, EBS1 and EBS2 A containing nucleotide integrase and the first sequence in the recognition site for the substrate Group II intron code binding to the element and the second sequence element Supplying the protein; and the nucleotide integrase is Breaks the double strand and inserts a Group II intron RNA at the top strand break. The nucleotide integrase and the double-stranded DNA substrate for sufficient time and temperature The step of reacting. The first sequence element of the recognition site is located upstream of the putative cleavage site. The IBS1 sequence and the IBS2 sequence. The first sequence element is about 10-12 base pairs. Of nucleotides. The second sequence element is a nucleotide of about 10 to 12 base pairs. And located downstream of the cleavage site (i.e., from position +1 to about +10, +11 or +12 Rank).   However, nucleotides that can be used to cleave both strands of a DNA substrate Glase is aI2 nucleotide integrase, al1 nucleotide integrase , LtrA nucleotide integrase, but are not limited thereto. aI2 Nucle The preferred recognition site for otide integrase is on its top strand, relative to the cleavage site. Relatively, C at -18, T at -15, T at -13, G at -16 or -19, T at +1 Contains T in +4 position and G in +6 position. DNA using aI2 nucleotide integrase To cleave both strands of the substrate, the substrate sequence is first examined and one strand is A set of nucleotides. Then, the aI2 intron RNA The EBS2 sequence is located downstream, adjacent to the substrate IBS2 sequence, i.e., T at position -13 No substantial homology to the sequence of 6 nucleotides and / or a The EBS1 sequence of the I2 intron RNA is adjacent to the substrate IBS1 sequence, Has no substantial homology to the 6 nucleotide sequence located downstream The EBS1 and EBS2 sequences of the Group II intron RNA at that time are described below. So that they are substantially homologous. aI2 nucleotide in Tegulase has a substrate whose top strand is A at position -21, G at position -19, C at position -18, G at position -16, and -1. Higher with T at position 5, T at position -13, T at position +1, T at position +4, and G at position +6 Cleaves both strands of the substrate with efficiency. aI2 is a substrate whose top strand is A at position -21, T, G at -19, C at -18, T at -17, G at -16, T at -15, T at -13, T at +1, With a T at +4 and a G at +6, both strands of the substrate are even more efficient. Disconnect. The top strand of the substrate is C at position +2, T at position +3, T at position +7, A at position +8, position +9 The cleavage is even higher with an A of T and a T at the +10 position.   al1 integrase has a top strand C at position -13 relative to the cleavage site, Relative to the cleavage site, T at position +1, T at position +2, T at position +3, T at position +4, A at position +5, +6 Used to cleave both strands of the DNA substrate, having a G at position, a T at position +7, and an A at position +8. Used. Preferably, the top strand of the double-stranded substrate is C at position -13, G at position -22, position -21. G, -19 in A, +1 in T, +2 in T, +3 in T, +4 in T, +5 in A, +6 in G, +7 It has a T at position and an A at position +8. Top strand in DNA substrate, G at position -22, G at position -21,- A in position 19, A in position -18, C in position -13, T in position +1, T in position +2, T in position +3, T in position +4, T in position +5, A in position +5 , G in +6, T in +7, A in +8, G in +9 and T in +10 High cutting efficiency. Substrate top strand further T at position -20, T at position -17, T at position -16, position -15 In addition, the cleavage is further enhanced when C is contained at position -14 and A at position -14.   In ltr nucleotide integrase, the top strand of the substrate is G at position -21 and the substrate is at position -20. A, C at +1, A at +2, T at +3, A at +4, T at +5, C at +6, A and +8 at +7 If it has a T at the position, it is used to cleave both strands of the double-stranded DNA substrate . For ltrA nucleotide integrase, the top strand is G at position -21, A at position -20, and position -19. T, G in -17, G in -15, C in +1, A in +2, T in +3, A in +4, T in +5, +6 With C at position, A at +7 and T at +8, both strands of the substrate are more efficiently cleaved. Refuse. The top chain of the substrate is C at position -22, C at position -18, T at position -16, A at position -14, -1 With A at position 3, T at position +9 and T at position +10, cleavage is further enhanced.   Another method is to cleave single-stranded nucleic acid substrates, i.e., single-stranded DNA or RNA. To bind the Group II intron RNA molecule to the cleavage site Use otide integrase. The method comprises the following steps: two on the substrate Can hybridize with IBS1 and IBS2, which are the intron RNA binding sequences of Group II intron RN with two hybridizing sequences EBS1 and EBS2 A and has non-conserved portions of the RT, X, and Zn domains Providing a nucleotide integrase comprising a Group II intron; And a step of reacting a substrate with nucleotide integrase. Group II b The EBS1 sequence of the intron RNA comprises 5-7 nucleotides and is in positions -1 to about -5 or -7. At least 80%, preferably 90%, more preferably the complete nucleotide Have the same sex. The EBS2 sequence of Group II intron RNA contains about 4-7 nucleotides. At least 80%, preferably 90%, of the nucleotides at about positions -6 to -14. %, More preferably complete homology. Preferably, the first nucleoside of EBS1 The preceding nucleotide adjacent to the tide is the nucleotide at position +1 in the sense strand. Is homologous to   The present invention also provides a method for determining whether a nucleic acid substrate contains a particular recognition site. Offer. This method is capable of cleaving a nucleic acid substrate having a specific recognition site. Providing a nucleic acid substrate; and providing the nucleic acid substrate with a nucleotide integrator. And assaying for cleavage of the substrate. Substrate cleavage Means that the substrate contains a specific recognition site. Fragmentation of nucleic acid substrates In addition to size change assays, group II introns on one strand of the nucleic acid substrate Cleavage can be detected by assaying for RNA incorporation or binding.   A wide range of temperatures is suitable for this method, but good results have been obtained at temperatures of about 30 ° C to about 42 ° C. At a reaction temperature, preferably from about 30C to about 37C. A suitable reaction medium is Na<sup>+</sup> Or K<sup>+</sup>At a concentration of about 0 to about 300 mM; preferably about 10 to about 300 mM. Containing about 200 mM KCl and a divalent cation, preferably magnesium or Cancer ions, more preferably magnesium ions at concentrations higher than 1 mM and lower than 100 mM. Including ON. Preferably, the divalent cation is at a concentration of about 5 to about 20 mM, more preferably About 10 to about 20 mM. The pH of the preferred medium is about 6.0 to 8.5, more preferably Is about 7.5-8.0.   In the above method, the single-stranded nucleic acid substrate, and the top strand of the double-stranded DNA substrate, It is thought to be cleaved by the removed group II intron RNA. Remove Cleavage catalyzed by the removed Group II intron RNA can be partial or complete. In addition, the cleavage site of the removed Group II intron RNA (ie, the top strand A reverse splicing reaction that results in an insertion at nucleotides -1 to +1). partially During insertion, the Group II intron RNA will have nucleotides + on the top strand of the cleavage site. Covalently bonded to 1. The bottom or antisense strand of the double-stranded DNA substrate It is believed that the protein is cleaved by the intron-encoded protein. Double-stranded DN A The bottom strand of the substrate is located about 9 to about 11 base pairs downstream of the cleavage site in the top strand, That is, it is cleaved at the site between nucleotide positions +9, +10 and +11.   Substrate DNA is purified using nucleotide integrase as an endonuclease Cleavage methods determine the presence and location of specific recognition sites on DNA substrates Is a useful analytical tool for In addition, either single-stranded or double-stranded DNA Nuclei to DNA substrates that occur when cleaved by nucleotide integrase Simultaneous insertion of acid molecules tags the cleavage site of the DNA substrate with a radiolabeled molecule This feature facilitates the identification of DNA substrates containing specific recognition sites. You. In addition, automatic binding of an RNA molecule to one strand of a double-stranded DNA substrate Through hybridization studies using probes that are complementary to the molecule, Allows identification of cleavage sites. Bound RNA, tagged with biotin-like molecules The molecule also makes it possible to affinity purify the broken strand.   Nucleotide integrators to cleave RNA or DNA substrates with recognition sites The use of a protease is useful for providing certain genes within a non-functional substrate. is there. Such methods also involve inserting the nucleic acid into the cleavage site, and thus the RNA component. Altering the properties of protein molecules encoded by proteins and substrates It is useful forNucleotide integrase   Nucleotide integrase is a ribonucleoprotein ("RNP") particle, And Group II intron coded RNA and Group II intron coded Contains proteins, the proteins of which bind to RNA. Preferably, the group II in Tron RNA is a Group II intron RNA that has been removed. As used herein, " `` Removed Group II intron RNA '' can be used in vitro or in vivo. Transcript of Group II intron DNA lacking adjacent exon sequences RNA The removed Group II intron RNA is either wild-type or modified. In vivo transcription and transfer from modified or unmodified Group II intron transcripts Mutated by pricing or by in vitro transcription and splicing Obtained from creatures. As used herein, "Group II intron coders" Protein is controlled by the Group II intron open reading frame. Is the protein to be loaded.   Group II introns are present in bacterial DNA and in organelles, especially fungi, yeast and Specific in mitochondria and plant chloroplast DNA Type of intron. Group II intron RNA molecules, in other words, RNA molecules encoded by loop II introns have similar secondary and tertiary The following structure is shared. Figure 2 shows the secondary structures of the al1 and aI2 intron RNAs, 2 shows the nucleotide sequence portions of wild-type al1 and aI2 intron RNA. Guru Group II intron RNA molecules typically have six domains. Group II a Domain IV of the intron RNA encodes the “Group II intron-encoded protein”. Contains the nucleotide sequence to be loaded.   Nucleotide integrase is, for example, a group II inn found in nature. A removed Group II intron RNA molecule having the same sequence as the tron RNA molecule I.e., wild-type group II intron RNA and glue found in nature. Group II intron R, which has a sequence different from that of Group II intron RNA NA, including modified and removed Group II intron RNA molecules. Break The altered and removed Group II intron RNA molecule is, for example, a Group II intron. Nucleotide base changes or additional nucleotides in the inner loop region of the tRNA Group II intron RNA molecules, preferably internal to domain IV Nucleotide changes in the loop region and the hybridizing region of domain 1. Includes Group II intron RNA molecules that have a functionalization. Group II intron RNA Eve Nucleotide integrase that has a nucleotide base change in the redisy region Compared to wild-type, typically for nucleotide DNA of integrase substrate With altered specificity.   The nucleotide integrase group II intron-encoded protein is X Domain and Zn domain. The X domain of the protein has a maturation activity (mat urase activity). The Zn domain of the protein is Zn²⁺Finger-like mochi Have Preferably, the Group II intron-encoded protein is further reversed Includes transcriptase domain. As used herein, Group II intron The encoded protein may contain additional amino acids at the N-terminus or C-terminus, or proteins. In internal regions of protein and wild-type group II intron-encoded proteins Includes modified Group II intron-encoded proteins with alterations. Guru Group II intron-encoded protein binds to the 3 'region of Group II intron RNA. It is believed that they match.   Nucleotide integrase is wild-type, mutant, or genetically engineered Provided in the form of RNP particles isolated from an organism. Nucleotide integrator Zetas may also be in the form of reconstituted RNP particles, isolated from reconstituted RNP particle preparations. Provided. Nucleotide integrase also eliminated exogenous synthetic Group II intron RNA, group II intron-encoded protein or RNA -Formed by combining with any of the protein complex preparations, Includes constituent RNP particles. Exogenous RNA is unmodified and modified Group II Includes both intronic RNA molecules. Preferably, the exogenous RNA is an in vitro transcript Or in vitro of unmodified or modified Group II introns It is a derivative of the transcript. For example, exogenous RNA can splice in vitro transcripts. Can be obtained. RNA-protein complex preparations Complex with an RNA molecule that is not a removed Group II RNA molecule that has a sequence to load And group II intron-encoded protein molecules. RNA-protein complex Group II intron-encoded proteins include ribosomal RNA molecules, mRNA molecules, Or a removed group that does not encode a Group II intron-encoded protein Binds to II intron RNA.   Nucleotide integrase can be obtained as purified RNP particles or Can be used as structured particles. Alternatively, the nucleotide integrase is RNP particles and reconstituted particles with nucleotide integrase activity And other RNP particles (eg, ribosomes), which can be used in partially purified preparations. You. This partially purified preparation is free of organelles.Preparation of nucleotide integrase   Nucleotide integrase is mitochondrial in wild-type or mutant yeast , Fungal mitochondria, plant mitochondria, chloroplast, proteoto Bacterium (Proteotobacterium) Azotobacter vinelandii, cyanobacterium (Cyanobacterium) Calothrix, and Escherichiacoli lactococcus l isolated from actis. The procedure for isolating the RNP particle preparation is based on the cell membrane of the organism and / or Or mechanically and / or enzymatically disrupting the cell wall. Fungi and In the case of plants and plants, purification can also include specific organelles (e.g., mitochondria or Chloroplast) from other cellular components by differential centrifugation and / or floating gradient. And then use a nonionic surfactant (eg, Nonidet P-40) Dissolving the organelles. The lysate of organelles and bacteria is then Centrifuge through sucrose cushion to prepare ribonucleoprotein (RNP) particles Get the product. RNP particles can be separated on a sucrose gradient or on a gel filtration column. Alternatively, it can be further purified by other types of chromatography.   Nucleotide integrase also converts RNA-protein complex preparations into exogenous Recombinants prepared by combining with excised Group II intron RNA Isolate from the constituent RNP particle preparation. The RNA-protein complex preparation is preferably From yeast, fungi, or bacteria, using the above protocol for RNP particles Isolated. The preparation of the RNA-protein complex is a Group II intron code With the excised Group II intron RNA having the protein coding sequence Group II intron-encoded protein molecules complexed with non-existent RNA molecules Including. Group II intron-encoded proteins of the RNA-protein complex preparation , Ribosomal RNA molecule, mRNA molecule or group II intron-encoded protein Binds to any of the excised Group II intron RNAs that do not encode quality .   Exogenous RNA is preferably obtained by in vitro transcription of a Group II intron. Or a synthetic molecule produced by in vitro transcription and self-splicing. You. Exogenous RNA can also be derived from cells or organelles in which the RNA occurs naturally, or From cells in which the altered intron is inserted and expressed, It can be made by isolating an intron RNA. Then, the exogenous RNA is converted to RNA- Add to the preparation containing the protein complex. Preferably, exogenous group II The intron RNA is first denatured. Exogenous RNA on ice to RNA-protein complex Add in.   In another embodiment, the isolated DNA fragment comprising a Group II intron DNA sequence Nucleotides are introduced into host cells to produce nucleotide integrases. Suitable DNA molecules include, for example, viral vectors, plasmids and linear DNA molecules. Is mentioned. After introducing the DNA molecule into the host cell, the introduced Group II The excised RNA molecule encoded by the long DNA sequence and the introduced group The protein molecule encoded by the II intron DNA sequence is formed in the cell As such, the Group II intron DNA sequence is expressed in a host cell. Cut out Group II intron RNA and group II intron-encoded protein Combine in a host cell to produce nucleotide integrase.   Preferably, the introduced DNA molecule also contains a promoter, more preferably a glue. And an inducible promoter operably linked to the pII intron DNA sequence. Preferred Alternatively, the DNA molecule may further comprise a nucleotide integrase (eg, an affinity Encoding a tag that facilitates isolation of the tag (such as a tag and / or an epitope tag) Contains the sequence Preferably, the tag sequence is an open reading frame sequence. At the 3 'or 5' end. Suitable tag sequences include, for example, a series of histidine residues. Group, herpes simplex glycoprotein D (ie, HSV antigen), or glutathione And S-transferase-encoding sequences. Typically, DNA molecules It also includes a nucleotide sequence encoding an origin of replication and a selectable marker. necessary Depending on the DNA molecule, encodes a molecule that regulates expression (eg, T7 lysozyme) Contains the sequence   DNA molecules containing Group II intron sequences can be converted by conventional methods (e.g., Cloning into vector and electroporation or CaCl_Two Introducing a vector into a host cell by conventional methods, such as a mediated transformation procedure To the host cells. The method used to introduce the DNA molecule is Specific host cells to be used. Suitable host cells are group II intron DNA A cell capable of expressing the array. Suitable host cells are, for example, heterologous or homogeneous cells. Fungal cells, yeast cells, mammalian cells and plant cells. Host cell genome And group II intron DNA sequences use different genetic codes If the group II intron DNA sequence has the codon corresponding to the genetic code of the host cell It is preferred to be modified to include Group II intron DNA sequences are representative Typically, they are newly constructed from synthetic oligonucleotides or have in vitro parts. Group II introns with different codons modified by site-directed mutagenesis Prepare the DNA sequence. Alternatively, the difference between the intron's genetic code and the host cell Correspond to the genetic code of the Group II intron to resolve differences A DNA sequence encoding a tRNA molecule is introduced into a host cell. If necessary, RNA Or factors that assist in protein folding or RNA or protein A DNA molecule containing a sequence that encodes a factor that inhibits solution is also introduced into the cell.   Next, the DNA sequence of the introduced DNA molecule is expressed in host cells, Provide the main cell. As used herein, the term “transformed cell” may further include A host cell that has been genetically engineered to contain DNA and is restricted to cancerous cells Not done. Next, RNP particles having nucleotide integrase activity are Isolated from transformed host cells.   Preferably, the nucleotide integrase is lysed by lysing the transformed cells (eg, For example, by disrupting the cell membrane of transformed cells mechanically and / or enzymatically. B) Isolate. The cell lysate is then fractionated into insoluble and soluble fractions . Preferably, the RNP particle preparation is isolated from the soluble fraction. RNP particle preparation Nucleotide integrase activity and ribosome, mRNA and tRNA molecules RNP particles, as well as other RNPs. Suitable for isolating RNP particle preparations A clever method, for example, is to centrifuge the soluble fraction through a sucrose cushion. Ceremony I can do it. Preferably, the RNP particles are obtained from an RNP particle preparation or from a soluble fraction ( For example, by separation on a sucrose gradient or gel filtration column, or other Further purification). For example, the desired RNP particle When a protein component is manipulated to include a tag, such as a series of histidine residues , RNP particles are further analyzed by affinity on a matrix that recognizes and binds tags. Can be purified from the RNP particle preparation by chromatography. For example, NiNTA S uperflow (Qiagen Chatsworth CA), Group II code protein, His₆Ta Suitable for the isolation of RNP particles with   The following method for nucleotide integrase preparation is described for illustrative purposes. , Is not intended to limit the scope of the invention.Prescription   RNP particle preparations of formulas 1 to 10 below and RNA-protein conjugate of formula 12 The body was transformed with the wild-type yeast strain Saccharomyces cerevisiae ID41-6 / 161 MATa adel (Hereinafter referred to as "161") and mitochondria and its derivatives. The mitochondria of wild-type yeast strain 161 are group II intron al1 and group Includes C0X1 gene containing II intron aI2.   The C0X1 gene in the mutant yeast strain lacks one of the group II introns. Or has a mutation in one of the Group II introns is there. Excised group II intron RNA molecule and group II intron The encoded protein is a group II present in wild-type and mutant yeast strains Derived from the introns al1 and aI2.   By convention, the intron composition of the C0X1 gene in different yeast strains is as follows: Shown in The superscript “+” indicates the presence of the al1 or aI2 intron, A superscript "0" indicates the absence of an al1 or aI2 intron and other superscripts Refers to a particular allele or mutation within the aI2 intron.Prescription 1   The RNP particle preparation was transferred to the mitochondrial of Saccharomyces cerevisiae wild-type yeast strain 161. A. The intron composition of the C0X1 gene of the wild type strain is 1⁺Two⁺It is. RNP The particle preparation contains RNP particles derived from the al1 intron and Excised al1 RNA bound to the encoded protein. Preparation In addition, RNP particles derived from the aI2 intron, and the excised aI2 RNA molecule and And an RNP particle containing the bound aI2-encoding protein.   To prepare the RNP particle preparation, yeast was added to 2% raffinose, 2% BactoPep 1 liter containing tone (from Difco), and 1% yeast extract (from Difco) 1.6-1.7 O.D. in liquid culture medium.₅₉₅Inoculated. Cell wall, yeast from ICN Digested with 40 mg of lytic enzyme. The cells are then broken mechanically using glass beads. Broken by crushing. Nuclei and cell debris were collected on a Beckman GSA rotor at 5,000 rpm for 5 minutes. Lysates were pelleted by centrifugation for minutes. Remove the supernatant and remove the Beckman Centrifuge at 13,000 rpm for 15 minutes with GSA rotor to obtain mitochondrial pellet Was. Mitochondria are added to a 44% sucrose solution layer, a 53% sucrose solution layer, and Overlay a suspension gradient consisting of a 65% sucrose solution layer and Beckman SW28 low And centrifuged at 27,000 rpm for 2 hours and 10 minutes. 53% / 44% mitochondria And 0.5M KCl, 50mM CaCl_Two, 25mM Tris-HCl (pH 7.5), 5m Suspend in buffer containing MDTT and add Nonidet P-40 to 1% final concentration To dissolve. The mitochondrial lysate was then transferred to a Beckman 50Ti rotor. At 50,000 rpm for 17 hours, 0.5 M KCl, 25 mM CaCl_Two, 25 mM Tris-HCl (pH 7.5), 5 Centrifuge through a 1.85M sucrose cushion in buffer containing mM DTT Thus, a pellet of RNP particles containing almost no mitochondrial protein was obtained. single The separated RNP particles were resuspended in 10 mM Tris-HCl (pH 8.0) and 1 mM DTT, Stored at ° C. The preparation may be repeatedly thawed and frozen before use.Formulation 1a Purified RNP particles   2.5 O.D. from formulation 1 in a volume of 150 μl.₂₆₀100 mM KCl, 2 mM gCl_Two 12 ml of 5-20 in a buffer consisting of 50 mM Tris-HCl (pH 7.5) and 5 mM DTT. % Linear sucrose gradient overlaid. The gradient was run in a SW41 rotor at 4 ° C for 40 Centrifugation was performed at 5,000 rpm for 5 hours. The gradient was fractionated into 35 fractions of approximately 0.325 ml . Fractions 12-20 contain purified RNP particles substantially free of ribosomal RNA. The position of the RNP particles in the gradient fraction was determined by Northern High using aI2 antisense RNA. Confirmed individually by hybridization. Ribosomal RNA in the gradient fraction small Subunit and large subunit locations were determined by fractionation on a 1% agarose gel. Confirmed individually by tidium bromide staining. Approximately 85% of ribosomal RNA It is found in the fraction without the RNP particles containing the reotide integrase.Formulation 2 mutant yeast strain 1 ⁰ 2 ^{+ t} RNP particle preparation from   RNP particles contain the excised aI2 RNA and aI2 encoding protein. Yeast strain 1⁰Two^{+ t} From Philip S. of the University of Texas Southwestern Medical Center. Perlman Expo And this is obtained from Moran et al., 1995, which is incorporated herein by reference.Mob ile Group II of Yeast Mitochondrial DNA Are Novel Site Specific Retroele mants , Mol. Cell Biol. 15, 2828-38. 1⁰Two^{+ t}Mutation The strain was constructed as follows: (i) aI2 intron from strain 161 was replaced with ClaI-BamHI plasmid. PBluescript KS from Stratagene⁺And clone pJVM4 to (Ii) pJVM4 was cut with ClaI and NdeI to remove the 5 ′ end of the insert; (Iii) Msp containing exon 1 and exon 2 of mitochondrial C0X1 gene Insert the I-NdeI fragment into the 5 'end of aI2 from yeast strain and C1036 lone The plasmid pJVM164 was obtained. In it, al1 is cut out from mitochondrial DNA The yeast strain C10361 △ one is described in Kennel et al., Which is incorporated herein by reference. , 1993,Reverse transcriptase activity associated with maturase-encoding group II intorons in yeast mitochondria , Described in Cell 73, 133-146 Prepared as follows. Change pJVMl64 to [rho⁰] Strain, and 1⁰Two^{+ t}Alleles, pairs By replacement, it was placed into intact mitochondrial DNA. This last step A non-reverted COXI mutant derived from mutant C1036 (strain 5B) (the construction of which was described in Kennel et al., 1993, ) And a glycerol-containing medium (GLY⁺) Breathing in And can grow and contain the transformed C0XI allele instead of the 5B allele. Achieved by selection for recombinant progeny.   Reactions and procedures for DNA cloning (eg, ligation, restriction Elementary digestion, bacterial transformation, DNA sequencing, etc.)Molecular cloning : a laboratory manual Second edition, Cold Spring Harbor Laboratory Press. Cold Sp ring Harbor. Performed according to standard techniques as described in N.Y. Yeast mitocon Doria transformation is also described in Belcher et al., 1994, Biolistic transformation of mit. ochondria in Saccharomyces cerevisiae, 101-115. N.-S. Yang and P.M. Chris tou (edit)Particle Bombardment Technology for Gene Transfer .0xford Unive Performed according to standard techniques as described in rsity Press, New York. RNP grain The offspring preparation is prepared as described in Formulation 1 with the mutant yeast strain 1⁰Two^{+ t}From mitochondria Produced.Formulation 3 mutant yeast strain 1 ^{+ t} 2 ⁰ RNP particle preparation from   Yeast strain 1^{+ t}Two⁰Is a derivative of wild-type yeast strain 161. Yeast strain 1^{+ t}Two⁰To Philip S. Per Obtained from Dr. lman, and this is Kennell et al., 1993. Cell 73, 133-146 Was prepared as follows. Yeast strain 1^{+ t}Two⁰Is wild-type 1 through mitochondrial transformation 61 S. diastaticus inserted into mtDNAC0X1A segment of the gene (this is aI2 Lack). Construction was started with plasmid pSH2. This plasmid contains pB Cloned into S + (Stratagene, La Jolla, CA) as a HpalI / EcoRI fragment All1 from wild-type 161 and some flanking sequences. That plus The plasmid was cut with ClaI near the 3 ′ end of al1 and BamHI at the downstream polylinker. And contains a gap containing the 3 'end of al1, most of E2, E3 and aI3S.diasta ticus Filled with ClaI / BamHI fragment from mitochondrial DNA (NRRL Y-2416) ,COX1Gene 1^{+ t}Two⁰The morphology was made. hybridCOX1-1^{+ t}Two⁰Including segment The plasmid MMC109 (biolistic transformation)MAT α ade2-10 1 ura3-52 kar1-1) Rho⁰Transformed into derivative. The resulting artificial petite (p etite) with the n161 / m161-5B strain andCOX1 1^{+ t}Two⁰ Gly containing allele⁺Recombinants were isolated. Hive successfully spliced The lid al1 allele is a single nucleotide change from C to T at position 2401 (Thr in the intron open reading frame₇₄₄From Leu to Different from the wild-type 161 allele. RNP particle preparation as in formulation 1 In the mutant yeast strain 1^{+ t}Two⁰From mitochondria. RNP particles were cut out Includes aI1 RNA molecule and al1-encoding protein.Formulation 4 mutant yeast strain 1 ⁰ 2 ^YAHH RNP particle preparation from   Yeast strain 1⁰Two^YAHHTo Phillip S. Obtained from Dr. Perlman, and this was mutagenized Using the pJVM164 plasmid, Moran et al., 1995, Mol. Cell Biol. 15, 2838-38 Was prepared as described in. Allele, exo of COX1 allele 4.4 kb MspI / extending from 217 nucleotides upstream of intron 1 through intron aI3 By oligonucleotide-directed mutagenesis of pJVM164 containing BamHI fragment Produced. Mutagenesis was performed by converting aI2 nucleotides 1473 to 1478 from GAT GAT to CAT CAT (D-49 Change from 1D-492 to HH). RNP particles contain mutated and excised aI2 RNA and AI2 coder with YADDYAHH mutation in reverse transcriptase domain of protein Contains protein. The RNP particle preparation was prepared using the mutant yeast strain 1 as in Formulation 1.⁰Two^YAHH From mitochondria.Formulation 5 mutant yeast strain 1 ⁰ 2 RNP particle from ^P714T   Mutant yeast strain 1⁰Two^P714TTo Philip S. Obtained from Dr. Perlman, and this is Kennell Et al., 1993,Cell 73, 133-146. Here this is n161 / m161-C1036 △ 1. RNP particles are mutated and excised aI2 Intron RNA molecule and missense mutation P in Zn domain₇₁₄AI2 with T Includes raw protein. The RNP particle preparation was prepared as described in Formulation 1 using the mutant yeast Stock 1⁰Two^P714TFrom mitochondria.Formulation 6 mutant yeast strain 1 ⁰ 2 RNP particle from ^HHVR   Mutant yeast strain 1⁰Two^HHVRTo Philip S. Obtained from Dr. Perlman, and this is mutagenesis Using the pJVM164 plasmid described above, M is incorporated herein by reference. oran et al., 1995, Mol. Cell Biol. 15, 2828-38. It produced using it. Alleles were constructed by site-directed mutagenesis of pJVM164. Was. The aI2 intron has the following changes: CATCACGTAAGA at positions 2208-2219 (sequence Number 9) to GCAGCTGCAGCT (H₇₃₆H₇₃₇V₇₃₈R₇₃₉To AAAA) and A₂₂₂₇From A To T (N₇₄₂I). This nucleotide integrase preparation was mutated and excised aI2 intron RNA, and aI2 code with missense mutation in HHVR motif Contains protein. The RNP particle preparation was transferred to mutant yeast strain 1⁰Two^HHVRMitochondria Produced.Formulation 7 mutant yeast strain 1 ⁰ 2 RNP particle from ^ΔConZn   Mutant yeast strain 1⁰Two^Δ_ConZnTo Philip S. Obtained from Dr. Perlman, and this is a mutation Using the induced pJVM164 plasmid, Moran et al., 1995, Mol. Cell Biol. 15, 28 Made as described in 28-38. Alleles are pJVM164 oligonucleotides Constructed by leotide-specific mutagenesis. aI2 intron has the following changes : TTATTTAGT from TAATAATAA (L₇₁₉F₇₂₀S₇₂₁To 0ch0ch0ch) It has become. RNP particles are mutated and excised aI2 intron RNA, and Zn domain Contains aI2-encoding protein, lacking most conserved motifs in . The RNP particle preparation was transferred to mutant yeast strain 1⁰Two^ΔConZnFrom mitochondria.Formulation 8 mutant yeast strain ^{1 0} 2 ^{CC /} 1 from RNP particles   Mutant yeast strain 1⁰Two^{CC / 1}To Phillip S. Obtained from Dr. Perlman, and this is a mutagenesis Using the generated pJVM164 plasmid, Moran et al., 1995 Mol. Cell Biol. 15,2828-3 8 using the nucleotide sequence as described. Allele, pJV M164 was constructed by site-directed mutagenesis. aI2 intron has the following changes Yes: 2172 ~ 2173 places TG to GC (C₇₂₄A), and 2180-2182 from TTG to AG C (I₇₂₆C₇₂₇From MA to MA). The RNP particles are mutated and excised RNA and first Zn⁺²Altered three amino acids in a finger-like motif Includes aI2-encoding protein with noic acid. RNP particle preparation was transformed into mutant yeast strain 1⁰Two^{CC / 1} From mitochondria.Formulation 9 mutant yeast strain 1 ^{0 2 CC /} 2-derived RNP particles   Mutant yeast strain 1⁰Two^{CC / 2}To Philip S. Obtained from Dr. Perlman, and this is a mutagenesis Using the generated pJVM164 plasmid, Moran et al., 1995 Mol. Cell Biol. 15,2828- Prepared as described in 38. Alleles are converted to pJVM164 site-specific Constructed by mutagenesis. The aI2 intron has the following changes: 2304-2305 From TG to GC (C₇₆₈A), and 2313-2314 are converted from TG to GC (C₇₇₁A). RNP particles consist of mutated and excised aI2 intron RNA, and Zn⁺²Finger Includes aI2-encoding protein with two altered amino acids in the motif No. The RNP particle preparation was transferred to mutant yeast strain 1⁰Two^{CC / 2}From mitochondria.Formulation 10 mutant yeast strain 1 ⁰ 2 ^H6 derived RNP particles   Philip S. A mutant yeast strain obtained from Dr. Perlman is described in Moran et al., 1995. As shown, the mutagenized plasmid pJVM164 was transformed into the mitochondrial of yeast strain GRF18. Prepared by importing Alleles, pJVM164 site-specific mutation Constructed by induction, and this comprises nucleotides 2357 and 2358 of the aI2 intron. Has the sequence CATCATCATCATCATCAT (SEQ ID NO: 10) inserted between RNP particles The preparation was prepared according to the protocol described above for Formulation 1, mutant yeast strain 1⁰Two^H6No mi Made from tochondria. RNP particles are mutated and excised aI2 intron RN A and has six histidines added to the C-terminus of the aI2-encoding protein Includes aI2 encoding protein.Formulation 11 RNP particles from Neurospora vehicle   Varkud strain of Neurospora vector (available from Fungal Genetics Stock Center Mitochondria from Lambowitz A.M. 1979, Preparation and analysis s of mitochondrial ribosome, Meth. Enzymol.59, 421-433 Was prepared. The viables are ground with glass beads and as described in Formulation 1. Mitochondria and RNP particles were isolated. RNP particles are cut coI int Includes proteins encoded by long RNA and coI intron.Formulation 12 Reconstituted RNP particle preparation   The reconstituted RNP particle preparation was purified from the exogenous, excised, in vitro, aI2 intron. RNA transcripts from mutant yeast strain 1⁰Two^ΔD5Of RNA-protein complex isolated from It was made by incubating with In this strain, aI2 Tron RNA lacks domain V and therefore is splicing defective. You. Mutant allele 1⁰Two^ΔD5To Philip S. Obtained from Dr. Perlman, and this is Final mating with yeast strain 1⁰Two⁺Described in Moran et al., 1995, except that Yeast strain 1⁺Two^ΔD5Was constructed using the same procedure used to create . The RNA-protein complex preparation was added in Formula 1 for the RNP particle preparation. 1 using the protocol described⁰Two^ΔD5From. 1⁰Two^ΔD5From mitochondria The isolated RNA-protein complex preparation does not contain the excised aI2 RNA, Contains aI2-encoding proteins related to other RNA molecules in the preparation.   Group II, where exogenous RNA has been inserted into the pBLUESCRIPT KS (+) plasmid Yeast mitochondrial C0X from the ClaI site of intron 1 (al1) to the BamHI site of aI3 Prepared by in vitro transcription of plasmid pJVM4 containing a fragment of one gene. Was. Plasmid pJVM4 contains the following C0X1 sequences: exon 2, aI2, exon 3 Part of the Rabini al1 and aI3 sequences. Sequence operates on T3 RNA polymerase promoter It is connected as possible. The sequence of exon 2 and exon 3 is derived from the RNA transcript Required for self-splicing of aI2 intron RNA. JVM4 to BstEII ( This cuts the 3 'end of exon 3) and then 5 μg of plasmid 0.300 ml of 40 mM Tris-HCl (pH 8.0), 25 mM NaCl, 8 mM MgCl_Two, 2mM spermidine, 5 mM DTT, 500 mM rNTP, obtained from 600 U RNasin and BRL from US Biochemical Incubate for 2 hours at 37 ° C in 300-750 U of T3 RNA polymerase. Transcripts were made. After incubation, RNA transcripts were extracted with phenol-CIA. Purified on a G-50 column, phenol-CIA extracted and ethanol precipitated. Next Then, transfer the RNA transcript to 40 mM Tris-HCl (pH 7.5), 100 mM MgCl_Two, 2M NH_Four40-45 ° C in Cl For 1 hour to allow the self-scanning of the aI2 intron RNA molecule from the RNA transcript. Spliced and the spliced product was obtained. Excised aI2 RNA Spliced product containing transcript, ligated lacking aI2 intron RNA Transcripts that have not been spliced and unspliced through a G-50 column And then phenol-CIA extraction and ethanol precipitation to yield exogenous RN. A offered. Then, 10 mM Tris-H was added to the exogenous RNA so that the final concentration was 1.0 μg / μl. Resuspended in Cl (pH 8.0), 1 mM EDTA.   To prepare a reconstituted RNA particle preparation, add 1 μl of exogenous RNA on ice to 0-10 2 μl for 1 min⁰2^ΔD5RNA-protein complex preparation (0.025 O.D.₂₆₀Unit) did. The preparation was used immediately.Formulation 13 Nucleotides containing group II intron RNA with altered EBS sequence Reconstituted RNP particles containing dosing integrase   Plasmid pJVM4 derivatives are used to prepare exogenous aI2 intron RNA molecules. Where the EBS1 and EBS2 sequences are different from the EBS sequence in the wild-type aI2 intron. different. pJM4 is downstream of the phage T3 promoter in pBluescript II KS (+) AI2 intron sequence and adjacent exons from wild-type yeast 161 cloned in Includes son sequence. Plasmids containing modified introns should be prepared with appropriate primers. It was derived from pJVM4 by the PCR mutagenesis used. Modified in all cases The region was sequenced to confirm that the correct and exogenous mutations were not present.   Plasmids pJVM4-aI1EBS1, pJVM4-aI1EBS2 and pJVM4-aI1EBS1 / EBS2 contain aI2R Including NA derivatives, where the EBS1 and / or EBS2 sequences were replaced with the sequence of al1. In each case, the 5 'and 3' portions of the exons were also The al1 sequence was changed to allow licensing. pJVM4-aI1EBS1 is 5'AGAA Has EBS positions 2985-2990 changed from GA to 5'CGTTGA; pJVM4-aI1EBS2 has 5'TCA EBS2 positions 2935-2940 changed from TTA to 5'ACTTTA; and pJVM4-aI1EBS1E BS2 ranked Nos. 2935-2940 and No. 2985-2990 from 5 'TCATTA to 5' ACAATT, respectively. And EBS1 and EBS2 changed from 5'AGAAAG to 5'CGTTGA. pJVM4-aI1EBS For 1 and pJV4-aI1 EBS1 / EBS2, 5 ′ of the pJVM4 insert consisting of the aI2 and E2 sequences The part was replaced with the last 24 bp of E1. For pJVM4-aI1EBS2, -24 to -7 position (GTCATGCTGTATTAATGA) was replaced with SEQ ID NO: 12 (ATYGGTAATTCACAATTAT), and a The I2 IBS1 sequence was left unchanged. For all three constructs, insert 3 The 'part was replaced by the first 15 bp of E2 instead of E3 and aI3.   pJVM4-EBS2-8G, pJVM4-EBS2-9T-10A, pJVM4-EBS2-11A, pJVM4-EBS2-12A and pVJM4-EBS2-13T (1) is a derivative of pJVM4 and the changes shown here are Have been introduced in different locations. pJVM4-EBS2-13T (2) has a second mutation (intron 293 Identical to pJVM4-EBS2-13T (1) except that it contains T to A) in position 2.   pJVM4-δ-C, pJVM4-δ-G, and pJVM9-δ-T are derivatives of pJVM4, Otides (2984th) replaced by C, G or T, respectively, Nucleotide is substituted at the α 'position of exon 3 for in vitro splicing ing.   Exogenous aI2 with modified EBS1 sequence and / or modified EBS2 sequence Intron transcripts are transferred to phage T3 polymerase and modified It was synthesized using Sumid. Synthetic transcripts are derived from yeast mitochondrial COX1 protein. Regions of the modified aI2 intron RNA and adjacent exons 2 and exons 3 regions. The synthetic transcript is self-spliced and The ising product is desalted through a G-50 column, extracted with phenol CIA, and ethanol And dissolved in TE (pH 8.0) at a final concentration of 1.0 μg / μl (0.52 μM).   The resulting modified ablated aI2 RNA molecule is formulated for RNP particle preparation Using the protocol described above in 1,⁰Two^ΔD5RNA-protein isolated from The conjugate preparation was individually mixed. This yeast mutant contains the domain of the aI2 intron. Has a deletion in the protein V and cannot splice aI2 RNA. This mutant is High expression of aI2 protein from unrice precursor mRNA. Therefore, RNA- Preparations of the protein complex contain higher amounts of aI2 protein.   For reconstitution, 1 μl of the spliced synthetic al2 transcript was added to 2 μl (0.025 OD_{26 0} Mix with the RNA protein complex preparation and incubate on ice for 0-10 minutes. Was.Prescription 14   The loop region of domain IV of group II intron RNA has been modified (ie, The nucleotide sequence of the loop region nucleotide sequence of domain IV is defined as RNP particle preparations containing RNP particles (different from the primer sequence) are prepared by two methods. You. First, oligonucleotide-directed mutagenesis of aI2 intron DNA The loop region of domain IV of aI2 intron RNA is coded by a well-known method. The nucleotide sequence to be changed. Then, the mutagenized aI2 intron D The NA is inserted into a vector such as a plasmid. Here, this DNA is Promote for RNase or SP6 RNA polymerase or T3 RNA polymerase Operably linked to an RNA polymerase promoter, such as The in vitro transcript of the selected Group II intron RNA was described above in Formulation 12. It is produced as follows. Exogenous RNA was then isolated as described for formulation 12. Combine with modified RNA-protein complexes to generate modified reconstituted RNP particle preparations Let it.   Alternatively, RNP particles in which the sequence in the loop region of the group II intron RNA has been modified The offspring preparation is prepared as described in Formulations 4-10 by site-specification of an organism such as yeast. Preparation of Biologically Derived RNP Particles by Dynamic Mutagenesis and as described in Formulation 1 Prepared by isolation of the product.Formulation 15 Preparation of RNP particles from genetically engineered cells   Lactococcus lactis Lactococcus conjugative element pRS0 Nucleotide containing truncated RNA encoded by one Ll.ltrB intron Co-integrase and the ORF LtrA of the Ll.ltrB intron. The protein is transferred to cells of the BLR (DE3) strain of the bacterium Escherichia coli (recA(With genotype) Was prepared by transformation with plasmid pETLtrA19. Plasmid pETLtrA 19 is located between the adjacent exons ltrBE1 and ltrBE2, Lactococcu s lactis contains the DNA sequence of the group II intron Ll.ltrB from Lactis. pETLtrA19 Also, DNA for T7 RNA polymerase promoter and T7 transcription terminator Contains the sequence. The sequence was such that the ORF sequence (SEQ ID NO: 6) in the Ll.ltrB intron was T7RN. Arranged in the plasmid so that it is under the control of the A polymerase promoter . The ORF of the Ll.ltrB intron encodes the protein ltrA. Present in pETLtrA19 The sequence of the Ll.ltrB intron and the adjacent exon sequence are shown in SEQ ID NO: 5. You. The amino acid sequence of the ltrA protein is shown in SEQ ID NO: 7. Domain IV is Coded by Otides 705-2572.   First, pETLtrA19 uses pLE12 obtained from Dr. Gary Dunny of the University of Minnesota as Hi by digestion with ndIII and isolating the restriction fragments on a 1% agarose gel. Prepared. Parts of exons ltrBE1 and ltrBE2 flanking the Ll.ltrB intron And a 2.8 kb HindIII fragment contained from the agarose gel. The strand overhang is made with the Klenow fragment of DNA polymerase I (Gibco BRL, Gaithersbur g, obtained from MD). The resulting fragment is deleted by XbaI. And ligated to plasmid pET-11a treated with Klenow fragment. pET-1 1a was obtained from Novagen, Madison, WI.   pETLtrA19 was used in the “Molecular Cloning A Laboratory Manual” at page 1-82 (1989). Conventional CaCl as described by Sambrook et al._TwoE. coli using a mediated transformation procedure. coli The cells were introduced. A single transformed colony is selected for plasmid selection. Luria-Bertani supplemented with picillin and tetracycline to select for BLR strains (LB) Selected on plates containing medium. Replace one or more colonies with ampicillin 2 ml of a filled LB medium was inoculated and grown overnight at 37 ° C. with shaking. This culture 1 ml of the mixture was inoculated into 100 ml of LB medium supplemented with ampicillin, and shaken at 37 ° C and 200 rpm. The OD of the culture with₅₉₅Was grown to 0.4. Then isopropyl -Β-D-thiogalactoside was added to the culture to a final concentration of 1 mM and incubated at room temperature. Lab continued for 3 hours. Thereafter, the whole culture is centrifuged at 2,200 xg, 4 ° C for 5 minutes. Collected by separation. Wash the bacterial pellet with 150 mM NaCl, and Finally, 1/20 volume of the first culture, 50 mM Tris, pH 7.5, lmM EDTA, 1 mM DTT and And 10% (v / v) glycerol (buffer A). Bacteria are frozen at -70 ° C Was.   Bacteria were thawed and frozen at -70 ° C three times to produce lysate. Next, 4 times the volume of 500 mM KCL, 50 mM CaCl_Two, 25 mM Tris, pH 7.5, and 5 mM DTT (H KCTD) to the lysate and allow the mixture to become viscous (ie, Sonicated. The lysate is centrifuged at 14,000 xg at 4 ° C for 15 minutes to obtain a soluble fraction. And an insoluble fraction. Next, 5 ml of the resulting supernatant (ie, soluble Load fraction) on a 1.85M sucrose cushion in HKCTD And centrifuged at 50,000 rpm for 17 hours at 4 ° C in a Ti 50 rotor (from Beckman). Was. The pellet containing the RNP particles is washed with 1 ml of water, then 25 μl of 10 mM Tris, p on ice. H 8.0, dissolved in 1 mM DTT. Centrifuge the insoluble substance at 15,000 xg at 4 ° C for 5 minutes. Removed by separation. The RNP particle product prepared according to this method contains the excised Ll.ltrB Contains intron RNA and ltrA protein.                               Preparation of substrate DNA   Sequence from the E2 / E3 junction of the yeast mitochondrial COX1 gene, yeast mitochondria Sequences from the E1 / E2 junction of the COX1 gene and putative Lactococcus lactis relax A labeled DNA group having a sequence derived from the E1 / E2 junction of the relaxase gene (ltrB) Quality from recombinant plasmids or by PCR or primer extension. Synthesized from a lignonucleotide template. Yeast mitochondrial COX1 gene The sequence of the substrate containing the E2 / E3 junction is shown in FIG. 3 as a wt sequence. FIG. 3 also shows The position of the mutation in the sequence is also identified. E1 / E2 connection of yeast mitochondrial COX1 gene The sequence of the substrate containing the junction is shown in FIG. FIG. 6 also shows the location of the mutation in this sequence. Is also identified. E1 / E2 conjugation of the putative Lactococcus lactis relaxase gene (ltrB) FIG. 8 shows the sequence of the substrate including the part. FIG. 8 also shows the location of the mutation in this sequence. Set. A DNA substrate labeled at the 5 'end of the antisense strand and a 5' PCR using 200 ng of the identified primer and 200 ng of unlabeled primer Generated from the code. Both primers are complementary to the sequence at the polylinker. Single-stranded DNA substrates were synthesized by end-labeling of the nucleotides. Short double-stranded DNA substrate The same segment was also prepared.   Excised aI2 intron RNA bound to aI2 protein, binds to al1 protein Excised al1 intron RNA or excised bound to ltrA protein Nucleotide integrase containing ltrA intron RNA cleaves DNA substrates The following examples of the method used to illustrate are by way of illustration only and are not intended to limit the scope of the invention. It is not intended to be limiting.Example 1 Including wild-type aI2 intron RNA and wild-type aI2-encoding protein Cleavage of double-stranded DNA substrate using nucleotide integrase   Formula 1 RNP particles 0.025 O.D.₂₆₀Units are yeast mitochondrial COX1 exon 2 and And a DNA substrate containing the WT sequence shown in FIG. 3 (E2E3). And reacted. The reaction was performed at 37 ° C in 100 mM KCl, 20 mM MgCl_TwoBuffer containing pH 7.5 Went inside. A part of the cleavage product is denatured with glyoxal and 1% agarose gel To determine the degree of cleavage of the top or sense strand at the E2 / E3 junction of the DNA substrate. I did. Another portion of the nucleic acid cleavage product is analyzed on a 6% denaturing polyacrylamide gel, The extent of cleavage in both strands of the double-stranded DNA substrate was determined. Dry the gel and autoradi Phosphor with Ographie or Molecular Dynamics Phosphorimager 445 Quantification by imaging (phosphorimaging) was performed.   The results indicate that wild-type bound to the aI2 intron-encoded protein from wild-type yeast Nucleotide integrase containing a truncated aI2 intron RNA from yeast Cleaves the top strand of the substrate having the wt target sequence at the position indicated by the arrow in FIG. Was shown. The results also show that the group II intron RNA was assembled at the sense strand cleavage site. It is shown that it will be incorporated. The results also indicate that the nucleotide integrase is a double-stranded DN The bottom strand or antisense strand of the A substrate is 10 base pairs below the cleavage site of the first strand. Flow It is shown that cutting is performed at the position.   Formula 1 RNP particles 0.025 O.D.₂₆₀The unit is the six different derivations of the wt DNA substrate of FIG. Reacted with body. Each derivative contains one point mutation in IBS2 of the wt sequence shown in FIG. I had. In derivatives, the nucleotides -7, -8, -9, -10, -11, -12, and -13 Each tide was converted to its complement. The reaction is performed similarly, and the cleavage product is Assayed on a 1% agarose gel as described above. The result is The ability of integlase to cleave double-stranded DNA substrates depends on each of EBS2 in the aI2 intron RNA. If the nucleotide and each nucleotide of the substrate IBS2 are not completely complementary, It showed a considerable decrease. The only exception is that the substrate mutates to +7 nucleotides. Happened when you have.   Formula 1 nucleotide integrase 0.025 O.D.₂₆₀Unit is from -14 of wt array -21 nucleotides at each position separately to form a mixture of incorrect nucleotides Reacted with a modified derivative of the wt DNA substrate of FIG. Therefore, nucleotide integration Gluase is used for 10 different substrates, each containing a mixture of three mutations at one site. And reacted. The reaction is performed as described above in Example 1 and the cleavage product is Oxylated and assayed on a 1% agarose gel. The result is the nucleotide in Tegulase has point mutations at positions -21, -20, -17, and -14 of the target sequence When the substrate is reacted with nucleotide integrase with the wt sequence shown in FIG. It has been shown to cut at levels ranging from 67% to 115% of the level achieved. Cutting Levels are most significantly reduced using substrates with point mutations at -15 and -18. Was. The level of cleavage that occurs with substrates having mutations at -15 and -18 9% of the cleavage obtained when the pide integrase reacted with the wt sequence shown in FIG. And 3%. Mutations at positions -16 and -19 have moderate effects and these Substrates containing less than 23% of the levels achieved with substrates having wt sequences Cleaved by nucleotide integrase at a level that is .   Formula 1 nucleotide integrase 0.025 O.D₂₆₀Unit is from +1 to +1 in wt array FIG. 3D in which the nucleotide at each position of 0 was separately changed to a mixture of three different bases. Reacted with derivatives of NA substrate. Therefore, nucleotide integrase is reduced to 30 Reacted with different substrates. Where each substrate is a mixture of three different nucleotides Having. The reaction was performed as described above in Example 1 and the cleavage product was Assay on acrylamide gel, nucleotides at these positions contain wt sequence Whether it was necessary for cleavage of the antisense strand of the substrate was determined. Glyoxyl And analyzed on a 1% agarose gel, the nucleotide changes at these positions Some influence on the ability of nucleotide integrase to cleave the top strand of the substrate Decided if there is. The results show that aI2 nucleotide integrase is at positions +1, +4, The substrate for the second strand having changes at positions +6 and Are 39%, 3%, respectively, of the levels achieved when reacting with the wt sequence shown in FIG. Cutting at 3% and 29% levels. Other positions (ie +2, +3 , +5, +7, +8, +9 and +10) nucleotide changes It had little effect on the ability of the substrate to cleave the second strand. The results also show that Nucleotide changes at each position are determined by nucleotide integrase Has little effect on the ability to cleave the strand.Comparative Example A   Formulation 1, 2, 4, 5 RNP particle preparation 0.025 O.D.₂₆₀The unit is the weight distribution shown in FIG. Reaction with 125 fmol (150,000 cpm) of an internally labeled DNA substrate having a row was performed for 20 minutes. Cutting To confirm the product, the product was glyoxylated and analyzed on a 1% agarose gel. Conclusion The result is a lack of excised aI2 intron RNA or an intron-encoded protein. Nucleotide integrase whose quality lacks the non-conserved portion of the Zn domain is a double-stranded DNA It showed no cleavage of the substrate nor adhesion to the RNA.Example 2 Cleavage of double-stranded DNA substrate using the reconstituted RNP particle preparation of Formula 12   The reconstituted RNP particle preparation of Formula 12 was generated from pE2E3 and 250 fmol (300,000 cpm) of a 142 bp DNA group with either 5 'end labeled sense strand The reaction was carried out at 37 ° C. for 20 minutes. To confirm cleavage of both strands of the substrate, The phenol was extracted with phenol CIA in the presence of 0.3 M NaOAc and 2 μg of single-stranded salmon sperm DNA, Subsequently, ethanol precipitation was performed. Reaction product is 6% polyacrylamide / 8M urea The gel was analyzed. The results show that the reconstructed particle preparation has the wild-type sequence shown in FIG. It was shown that both strands of the containing double-stranded DNA substrate were cleaved. A similar result, that is, both Strand cleavage was performed by incubating the 5 'end-labeled substrate with the RNP particle preparation of Formula 10 If you got.Example 3 Contains modified aI2 intron RNA and aI2 encoded protein Cleavage of double-stranded DNA substrate using nucleotide integrase   Formula 13 RNP particle preparation 0.025 O.D.₂₆₀Unit (here, aI2 Group II Was changed to the EBS1 sequence of the al1 intron RNA). DNA substrates and derivatives thereof (here, nucleotides at positions -1 to -6 are simultaneously (It was changed to 5'TTAATG, which is the IBS1 sequence of the wt sequence of l1 nucleotide integrase.) And reacted. Perform the reaction and analyze the cleavage products as described in Example 1. did. The result is aI2 nucleoside containing group II intron RNA with modified EBS1 Tide integrase was unable to cleave substrates with wt sequences, but from positions -1 to -6 Can cleave a substrate that is complementary to the modified EBS1.Example 4 Wild-type or modified aI2 intron RNA and aI2-encoding protein Cleavage of substrate using nucleotide integrase containing.   Formula 1 RNP particles 0.025 O.D.₂₆₀The unit is the three different inductions of the DNA wt substrate of FIG. Reacted with body. Each derivative contained a single point mutation. In derivatives, +1 nuku Leotide was changed to either C, G, or A. Derivatives are also added immediately before EBS1 Contains an aI2 intron RNA in which the nucleotides are any of A, G, C, or T. With nucleotide integrase. As described in Example 1, Reactions were performed and cleavage products were assayed on a 1% agarose gel. The result is a +1 If the nucleotide is complementary to the nucleotide immediately before EBS1 in the aI2 intron RNA In this case, cleavage of the top strand is enhanced, the target sequence has a G at position +1 and the intron RN If A has a purine nucleotide (A or G) at the δ position, cleavage of the sense strand is It showed a large decrease.Example 5 contains al1 intron RNA and al1 intron encoded protein Cleavage of double-stranded DNA substrate using nucleotide integrase.   Either the wt sequence or the altered sequence having one of the 11 single point mutations shown in FIG. Was reacted with the RNP particle preparation of Formula 3. Of each reaction For this, 1.5 nM (150,000 cpm) of double-stranded DNA substrate was added to the RNP particle preparation 0.025 O.D.₂₆₀unit And 10 μl of 50 mM Tris pH 7.5, 5 mM KCl, 10 mM MgCl_Two, 5 mM DTT. Anti The reaction mixture was incubated at 37 ° C. for 20 minutes. 70 μl of 28.6 mM EDTA, 0.15 mg / m The reaction was stopped by adding ltRNA. Extract nucleic acid with phenol Tanol precipitated, glyoxylated and analyzed on a 1% agarose gel.   The results show that the nucleotide integrase in Formula 3 is at positions -23, -20, -17 and -16 Substrate DNAs having mutations at positions -15, -15, and -14 have the wt sequence shown in FIG. It was shown to cleave with the same efficiency as the substrate. G (-22), G (-21), A (-19), and A ( Mutation at position -18) can reduce the efficiency of the cleavage by 75% to 25% of the cleavage that occurs with the wt sequence. The rate was reduced. The most important nucleotide appears to be the C at position -13. This Mutation at the position reduced substrate cleavage to less than 1% of cleavage occurring with the wt sequence.Example 6 Ll.ltrB Intron RNA and Ll.ltrB Intron Encoding Protein Of substrates using nucleotide integrase containing   either the wt sequence or the altered wt sequence having one of the 11 single point mutations shown in FIG. The double-stranded DNA substrate containing this was reacted with the RNP particle preparation of Formulation 15. Point mutation Occurs at positions -23 to -13 of the wt sequence. 1.5 nM for each reaction The double-stranded DNA substrate was prepared using the RNP particle preparation 0.025 O.D.₂₆₀Unit and 10 μl of 50 mM Tris pH 7.5 , 10mM KCl, 10mM MgCl_Two, 5 mM DTT. Reaction mixture at 37 ° C for 20 minutes Incubated. By adding 70 μl of 28.6 mM EDTA, 0.15 mg / ml tRNA To stop the reaction. Nucleic acid is extracted with phenol, ethanol precipitated, and It was silylated and analyzed on a 1% agarose gel.   The results show that the nucleotide integrase in formulation 15 has the wt sequence shown in FIG. C (-22), C (-18), A (-l) at about 80% of the level achieved by the substrate It was shown that the substrate DNA having a mutation at position 4) was cleaved. G (-21) and A (- 20), substrate with a point mutation at position T (-19) is achieved using a substrate with a wt sequence It was cut at a level of less than about 40% of the level achieved.Example 7 Cleavage of double-stranded DNA substrate using purified RNP particles   Containing yeast mitochondrial COX1 exons 2 and 3 (E2E3) and FIG. Formulated with 125 fmol (150,000 cpm) of internally labeled substrate containing WT sequence shown in sucrose of 1a Incubated with 10 μl of each fraction obtained from the gradient. Taking into account the composition of the fraction , 20 μl of the final reaction medium is 100 mM KCl, 20 mM MgCl_Two, 50 mM Tris-HCl, pH 7.5, and And 5 mM DTT. Following a reaction at 37 ° C. for 20 minutes, 30 μl of water, 5 μl of 0.3 M NaO Ac and 5 μg of tRNA were added to the fractions. The reaction product is extracted with phenol and ethanol Precipitated, glyoxylated, separated on a 1% agarose gel and dried The gel was analyzed by autoradiography. The result is the purified RNP particles of Formula 1a Is used to cleave both strands of the double-stranded DNA substrate and to insert the aI2 intron RNA into the cleavage site. It was shown to be useful.Example 8 Cleavage of both strands of double-stranded DNA substrate and cleavage site of antisense strand of cDNA Bonding to   RNP particles of formula 1, 2, 4, 5, 6, 7, 8, 9 0.025 O.D.₂₆₀The unit is the WT sequence shown in FIG. Incubated with 250 fmol (300,000 cpm) of the containing 142 base pair DNA substrate. DNA incubation The product was analyzed on a 6% polyacrylamide / 8M urea gel.   The radiolabeled band corresponding to the 5 'fragment With a substrate labeled at the 5 ′ end of either the top or bottom strand of the DNA substrate It was detected when incubation was carried out. This is because these particles are Indicates that both strands were cut. The RNP particles of Formulation 1 have a top chain of exon 2-E Cut exactly at the Kisone 3 junction. The RNP particles of Formulations 1 and 2 contain top chains or Cuts the bottom or antisense strand 10 base pairs downstream from the sense strand cleavage site. Was. Formula 1 treated or boiled with protease K or RNase A RNP particles did not break either strand.   The radiolabeled band also showed that the RNP particles of Formula 4 Detected when incubated with DNA substrate 5 'end labeled with either strand Was issued. This is because this nucleotide integrase binds both strands of the DNA substrate. Indicates disconnection. The RNP particles in Formula 4 were modified and excised aI2 RNA, and And aI2-encoding proteins lacking detectable reverse transcriptase activity. place The degree of cleavage of the RNP particles in Method 4 will be compared to that of the RNP particle preparation of Formulation 1. Although reduced, the endonuclease activity of the RNA is opposite to that of the aI2-encoded protein. It is present even in the absence of transcriptase activity.   The radiolabeled band shows the RNP particles of Formulation 5 on either the top or bottom strand. Detected when incubated with a DNA substrate labeled at the 5 'end . O.D.₂₆₀Quantification normalized by either or soluble aI2 reverse transcriptase activity In the assay, the activity of cleavage of the top and bottom chains by the RNP particles of formulation 5 Were 6% and 25% of the activity of the RNP particles of Formula 1, respectively.   The radiolabeled band corresponding to the 5 'fragment is labeled at the 5' end of the top strand. Was detected when the DNA substrate was incubated with the RNP particles of formula 6, The band corresponding to the 5 ′ fragment of the top strand is the RNP particle of formulation 6 with the 5 ′ fragment of the bottom strand. 'Not detected when incubated with end-labeled DNA substrate Was. The RNP particles of Formula 6 were modified, excised aI2 intron RNA, and putative Includes aI2-encoding protein with a change in one of the endonuclease motifs No. Similar results were obtained for the RNP particles of Formula 7. Formula 7 is modified AI2 intron RNA removed and aI without conserved portion of Zn domain Includes 2 encoded proteins. Similarly, the RNP particles of Formulations 8 and 9 (each modified , Excised aI2 intron RNA, and Zn²⁺Mutation in the motif-like motif aI2 encoding protein) cleaved the sense strand of the DNA substrate, The antisense strand was not cleaved. For RNP particles in Formulations 6, 7, 8, and 9 For example, the level of sense strand breaks may be determined by RNA-DNA production detected on agarose gels. It was proportional to the quantity of things. These findings indicate that the antisense strand of the aI2-encoded protein 2 shows that endonuclease activity is related to the Zn domain.   The radiolabeled band corresponding to the 5 'fragment was used for the preparation of reconstituted RNP particles in Formula 12. Is labeled at the 5 'end of either the sense or antisense strand of the DNA substrate. Was detected when incubated with the substrate. These results are reconstructed Establish that the RNP particle preparation cleaves both strands of the DNA substrate.   Therefore, the nucleotide RNA integrase catalytic RNA molecule and intron code Both proteins are required to cleave both strands of double-stranded DNA. Int Certain modifications of the Zn and X domains of the Ron-encoding protein are Breaks down the antisense strand of otide integrase.   Formulations 1, 2, 4, and 5 RNP particle preparation 0.025 O.D.₂₆₀Unit: 10 μl of reaction medium Inside, 1 μg of the plasmid containing the wild type sequence shown in FIG. 4 was combined. Reaction medium The body contains 0.2 mM each of dATP, dGTP, and dTTP, 10 μCi [α-³²P] -dCTP (3,000Ci / mmo l; DuPont NEN, Boston MA), 100 mM KCl, and 5 mM dithiothreitol, 2 mM MgC l_Two, And 50 mM Tris-HCl, pH 8.5. The reaction involves adding the RNP preparation. Incubate at 37 ° C for 10 minutes, add 0.2 mM dCTP, add another 10 Chase for minutes. After a follow-up period, the reaction was allowed to proceed with 0.3 M sodium acetate, pH 7.8 and And 5 μg of E. phenol in the presence of E. coli tRNA carrier (Sigma, St. Louis, MO) CIA (phenol-chloroform-isoamyl alcohol; 25: 24: 1) extraction And the reaction was terminated. The product was precipitated twice with ethanol and 90 mM Tris-Ho 1% agarose containing acid, pH 8.3, 2mM EDTA and 0.05% ethidium bromide Dissolve in gel. The results show that the RNP particles of Formulations 1 and 2 show that the cleaved DNA substrate It has been shown to catalyze the formation of DNA molecules above. The results also show that the resected glue Group II ints lacking intron II RNA or reverse transcriptase domains Nucleotide integrase containing Ron-encoded protein is truncated It has also been shown not to catalyze the formation of cDNA molecules on the strand.Cleavage of single-stranded DNA   AI2 nucleotide containing the excised aI2RNA and aI2-encoding protein Integrase is complementary to the EBS1 and EBS2 sequences of the wild-type aI2 intron RNA. It was used to cut single-stranded DNA containing the IBS2 and IBS1 sequences. +1 to + If three nucleotides of 3 can base pair with the three nucleotides immediately upstream of EBS1, The response was greatly improved. Cleavage of substrate by nucleotide integrase and The most preferred reaction conditions for insertion of the intron RNA into the cleavage site are 100 mM KCl , 20mM MgCl_TwoPH 7.5, 5 mM DTT, 37 ° C.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), AU, CA, JP (72) Inventor Guo, Huatao             United States Texas 78741, Oh             Stin, Burton Drive Number             156 1845 (72) More, George             United States Texas 78751, Oh             Stin, Avenue Sea 4514 (72) Inventor Beer, Clifford J.             United States Ohio 43202, Coro             Mumbus, King Avenue 58

Claims (1)

[Claims] 1. A method for cleaving a double-stranded DNA substrate at a cleavage site, wherein the substrate has a recognition site. And the method comprises the following steps: (A) providing a nucleotide integrase comprising:   (I) The first intron RNA binding sequence that binds to the sequence of one strand of the DNA substrate A first hybridizable sequence capable of hybridizing and said one of said substrates Second hybridization capable of hybridizing to a second RNA binding sequence on the strand A group II intron RNA having an intron sequence; and   (Ii) at least one nucleotide of the first sequence element at the recognition site of the substrate A group II intron-encoding protein capable of binding to an otide; Said group II intron-encoding protein bound to an intron RNA; (B) allowing the nucleotide integrase to cleave one strand of the DNA substrate; The substrate and the nucleus to insert the group II intron RNA into the cleavage site Reacting reotide integrase, A method comprising: 2. The first hybridization sequence and the first intron RNA binding sequence There is at least 80% complementarity with the column and the second hybridisation At least 80% complement between the sequence and the second intron RNA binding sequence. The method of claim 1, wherein 3. The group II intron RNA is a δ'nucleotide on one strand of the substrate. Further comprising a δ nucleotide that is complementary to the cleavage site, wherein the δ ′ nucleotide is the cleavage site. The method of claim 1, wherein the method is in the +1 position with respect to 4. Wherein said Group II intron RNA is wild-type or modified aI2 intron RN A and the group II intron-encoded protein is aI2 intron coding 2. The method of claim 1, wherein the protein is a protein. 5. One strand of the substrate has a T at position -13 with respect to the cleavage site, T at position -15, C at position -18 relative to the cleavage site, and position -16 relative to the cleavage site Or the G at position -19. 6. The single-stranded substrate is composed of G at position -19, C at position -18, and G at position -16 corresponding to the cleavage site. 5. The method of claim 4, comprising a T at position -15, and a T at position -13. 7. Wherein said group II intron RNA is wild-type, or a modified aI1 intron RNA, wherein the group II encoded protein is coded by an aI1 intron. 2. The method of claim 1, which is a protein to be loaded. 8. Wherein one strand of the substrate has a C at the -13 site relative to the cleavage site. Item 8. The method according to Item 7. 9. One strand of the substrate has a G at position -22, a G at position -21, 8. The method of claim 7, comprising A at position, A at position -18, and C at position -13. 10. The nucleotide integrase is wild-type or modified, Ll.ltrB Including a protein encoded by a Tron RNA and the Ll.ltrB intron. The method of claim 1. 11. One strand of the substrate has a G at position -21 and a position at position -20 relative to the cleavage site. 11. The method of claim 10, comprising A. 12. One strand of the substrate has a G at position -21, an A at position -20, -1 relative to the cleavage site. 12. The method of claim 11, comprising T at position 9, G at position -17, and G at position -15. 13. A method for cleaving a single-stranded nucleic acid substrate at a cleavage site, comprising the following steps: (A) providing the following nucleotide integrase;   (I) a nucleic acid capable of hybridizing with a first intron RNA binding sequence of the nucleic acid substrate; A hybridizing sequence and an intron RNA binding sequence of the nucleic acid substrate A Group II intron RNA having a second hybridizable sequence capable of being hybridized; And   (Ii) a group II intron code bound to the group II intron RNA Protein; and (B) causing the nucleotide integrase to cleave the nucleic acid substrate, and In order to insert the group II intron RNA at the Reacting glase, a process, A method comprising: 14． 14. The method according to claim 13, wherein said substrate is RNA. 15. 14. The method according to claim 13, wherein said substrate is DNA. 16. 14. The method of claim 13, wherein the nucleotide integrase is under: (A) wild-type or modified aI2 intron RNA and aI2 intron coding Deprotein; (B) wild-type or modified aI1 intron RNA or aI1 intron Deprotein; and (C) wild-type or modified L1.ltrB intron RNA and L1.ltrB int Ron encoded protein, A method selected from the group consisting of 17． In a method that cleaves both strands of a double-stranded DNA substrate, the following steps: (A) providing a nucleotide integrase comprising:   (I) hybridizing to the first intron RNA binding sequence of one strand of the DNA substrate A first hybridizing sequence, and a second int of the one strand of the DNA substrate. Having a second hybridizing sequence capable of hybridizing with a long RNA binding sequence, Group II intron RNA; and   (Ii) at least one nucleotide of the first sequence element of the substrate; And a group capable of binding to at least one nucleotide of the second sequence element A II intron encoded protein, wherein said group II intron encoded protein is A protein is bound to the group II intron RNA; (C) the nucleotide integrase cleaves the DNA substrates of both strands, Sufficient time to be able to insert the group II intron RNA at the two strand breaks. Reacting the substrate and the nucleotide integrase at a temperature and A method comprising: 18. Wherein the nucleotide integrase is selected from the group consisting of: Item 18. The method according to Item 17, (A) wild-type or modified aI2 intron RNA and aI2 intron coding Deprotein; (B) wild-type or modified al1 intron RNA and al1 intron RNA Deprotein; and (C) wild-type or modified L1.ltrB intron RNA and L1.ltrB int Ron encoded protein. 19. The first hybridization sequence and the first intron RNA binding There is at least 80% complementarity with the sequence and the second hybridizer At least 80% of the phase between the alternative sequence and the second intron RNA binding sequence. 18. The method of claim 17, wherein the method is complementary. 20. The group II intron RNA is wild-type or modified aI2 intron RN A, wherein the group II intron-encoded protein is aI2 intron-encoded A protein and one chain of the substrate is C at position -18, T at position -15, -13 At position -13, G at position -13 or -16, T at position +1, T at position +4, and G at position +4. The method according to claim 17. 21. Wherein said group II intron RNA is wild-type or modified al1 intron RN A: wherein the group II intron-encoded protein is the al1 intron And one chain of the substrate is at position -13. C, T at + 1st place, T at + 2nd place, T at + 3rd place, T at + 4th place, A at + 5th place, G at + 6th place, T at + 7th place 18. The method of claim 17, comprising A at position +8. 22. Group II intron RNA is wild-type or modified LI.ItrB intron RN A and group II intron-encoded proteins are coded by the LI.ItrB intron. Where the top chain substrate is G at position -21, A at position -20, C at +1, A at +2, T at +3, A at +4, T at +5, C at +6, A at +7, 18. The method of claim 17, comprising T at position +8. 23. The Group II intron-encoding protein contains a reverse transcriptase domain. And the nucleotide integrase and the substrate comprise a cDNA molecule of the DN A dATP, dGTP, dTTP and dCTP as formed at the cleavage site of the other strand of the A substrate 18. The method according to claim 17, wherein the reaction is performed in a reaction mixture comprising: 24. A method for detecting the presence of a nucleotide recognition site in a nucleic acid substrate, comprising: Process of: (A) A nucleotide integrase capable of cleaving a nucleic acid substrate having a recognition site is provided. Providing; (B) reacting the nucleotide integrase with the nucleic acid substrate; (C) assaying for cleavage of the nucleic acid substrate, wherein the cleavage is performed on the nucleic acid substrate The process, which is an indicator of the presence of the recognition site in A method comprising: