TW200413528A - RNA-cleaving DNA enzymes with altered regio-or enantioselectivity - Google Patents

Abstract

The present invention provides catalytic single-stranded DNA molecules, and methods using the same, that have site-specific endonuclease activity that is specific for a cleavage site in a substrate nucleic acid sequence, that includes a non-naturally occurring single-stranded ribonucleic acid. The catalytic DNA molecule includes one or more loop regions and one or more binding regions, wherein the binding regions bind to complementary sequences of the substrate nucleic acid sequence. These non-naturally occurring single-stranded nucleic acids include a 2', 5' linked residue or an L-enantiomer residue.

Description

3528 发明 Description of the invention: [Technical field to which the invention belongs] This month 疋 related to nucleic acid enzymes or catalytic (enzymatic) ^ ^ eight molecules, which ^ ° 彳 unnaturally generated cardiac nuclear acid, especially ribose nuclear acid . [Prior art] _ "9 important characteristics are its catalytic rate enhancement effect, chemical transformation of I # r ^%, <specificity. There are many kinds of specificity, including the special ~ and Bailey's Special regioisomers and special stereostructures. Whether it is a naturally occurring enzyme composed of protein or nucleic acid, one can show various types of selectivity due to: complex structure-structure. Even if it is Simple peptides or nucleotides can operate I-3 with a high degree of specificity. The ability to obtain novel proteins and nucleic acid enzymes through direct long-distance opening has been developed to perform regional- or stereoselective chemical reactions. Artificial enzyme ㈠. The protein enzymes issued have been found to be industrially useful as chiral selective (dmOSelectlve) catalysts 8. Has been catalyzed Diener-Adeyi-Guoli (enanit〇menCeXCeSS 'denting into ee ) &gt; 95% (see 9) to operate. When the same RNA enzyme is prepared from L_ instead of 〇_nucleo # acid, it can of course produce relative enantiomeric products with the same ee value. No longer, Nucleic acid enzymes can be manipulated in a region-specific manner . For example, # 颏 RNA ligase ribozyme can selectively catalyze the formation of 3,5,5, and r'J '' armate diglyase to form 1G'U. Hammerhead ribozyme can cleave RNAt3, 5, Instead of 2, the phosphodiester is linked to 12. Delta Hepatitis Virus ribozyme can be differentiated, ^ ^ natural J,)-linked, but can also cut wJ · phosphate di <S7851 200413528

For the specific cutting of non-natural polynuclear nucleus, such as including 2 and 2 dicarboxylic acid, there are still some advantages in this == prime-can be used in diagnostic analysis =; ^ reduction level naturally generated by non-specific cutting Polynuclear acid. [Summary of the Invention] A new type of nucleic acid enzyme with region- and antipodal specificity produced by two Γ 疋 can be used to isolate novel and enzyme-group-cuttable 5 | _ linked p_D marriages using in-house expansion methods. The cleavage permits the cleaving of the 3 ', 5' · linked β-ribonucleotide (Figure 1A). DNA enzymes can be found by spreading in a test tube, starting from separate groups of DNA molecules of any sequence ~ 1 () 15. Each group can generate a DNA enzyme related to Mg2 + _, which performs the target reaction. Both groups of enzymes can be made &amp; to operate with multiple-enzyme turnover on separate nucleic acid substrates. It can cleave the enzyme DNA group of 2,5, _dipic acid diacetate, which exhibits a rate-enhancing effect of about 2G, and deletes the DNA enzyme group that can cleave L_ribonucleic acid without catalysis and shows About 600 times &amp; catalytic rate enhancement. The former operates with a regional specificity of about 6000-fold, while the latter operates about 50-fold with enantioselectivity. In one aspect, the present invention proposes a catalytic single-stranded DNA molecule that has position-specific endonuclease activity and is specific for the cleavage position in the acceptor nucleic acid sequence, and the cleavage position includes non-naturally occurring Single-stranded RNA, or a combination thereof. Catalytic DNA molecules include more than one loop region and more than one binding region, where the binding region binds the complementary sequence of the acceptor nucleic acid sequence. 87851 200413528 Catalytic single-stranded DNA molecules have position-specific endonuclease activity for the acceptor nucleic acid sequence, which can include various non-naturally-generated single-stranded nucleic acids at the cleavage position. Preferred examples of these non-naturally occurring single-stranded nucleic acids include 2 ', 5'-linked residues, such as 2', 5'-linked adenine riboic acid or guanine ribonucleotide ribonucleotide Residues, or L-enantiomeric residues, such as the L-enantiomer 3f, 5'-linked adenine nucleotide residues. In one aspect, the present invention provides a catalytic single-stranded DNA molecule that cleaves a acceptor nucleic acid sequence, including a 2 ', 5'-linked ribonucleic acid, preferably a 2', 5f-linked adenine core Gallic acid or guanine nucleotide ribonucleotide residues. The catalytic DNA molecule of this aspect of the present invention has a catalytic function site and a confirmation function site. The catalytic functional site includes a downstream region including the sequence S'-XiXaACTCGGAGXsJ, (SEQ ID NO: 28), which can form a loop zone; a central stem axis region, which is adjacent to the downstream loop zone 5 'and has a 5'-ZιZ2Z3Z4_3 'The sequence, and an upstream region adjacent to the central stem axis region 5', can form a loop with the sequence 5, -GGGA-3 ,. Xι is the required cytosine nucleoside residue, &amp; is the cytosine nucleoside or thymidine core residue, and X3 can bind the complementary nucleotide on the acceptor nucleic acid sequence, which is two upstream from the cutting position Nucleotides. A can bind complementary nucleotides on the acceptor nucleic acid sequence, which is immediately downstream of the cleavage site. In a preferred embodiment, the &apos; heart is a cytosine nuclear residue, and &amp; is a guanidine residue. Confirm that the functional part includes an upstream side connection area and a downstream side connection area. The upstream flank region is adjacent to 5 of the upstream ring zone, and the downstream flank region is adjacent to 3 of the downstream ring zone. In various specific examples, individual nucleotides in the flanking region can be bound to the flanking region of the acceptor nucleic acid sequence. 87851 -8-200413528 In some specific examples of this aspect of the invention, the downstream loop zone has the sequence 歹 ij of 5, -CCACTCGGAG-3 '(SEQ ID NO: 22). In some specific examples of this aspect of the invention, the upstream loop zone has the sequence 5'-YGGAA ^, where Y is 0 to 5 nucleotides. In these specific examples, Y can have essentially any nucleotide sequence of 0 to 5 nucleotides. For example, Y may include the sequence 5′-TTA-3, for example, Y may be 5, -GTTTA-3, (SEQ ID NO: 19), Si-GCTTA. ^ (SEQ ID NO: 20), S ′ -GTTA-S1 (SEQ ID NO: 21). In another aspect as illustrated in FIG. 14B, the present invention provides a catalytic single strand DNA molecule that can cleave the acceptor nucleic acid sequence on the L-ribonucleic acid residue, preferably the linked L-adenosine Ribonucleotide ribonucleotide residues. In this aspect, the catalytic DNA molecule includes a confirmation functional site and a catalytic functional site, which can form a loop including a nucleic acid sequence of 5LXA2X3GX4X5X6X7 GACX8X9-3, (SEQ ID NO. · 29). XiT binds to a complementary nucleotide on the acceptor nucleic acid sequence, which can be adjacent to the downstream of the cleavage site on the acceptor nucleic acid sequence. X2 is the thymidine or guanidine residue, X3 is the cytosine core or guanidine residue, and X4 is Cytocarcinoma nucleus or thymic nucleus residue, X 5 is cytosine or thymidine, X6 is cytosine or thymic nucleus, X7 is gland: purine nucleus or guanidine residue, X8 It is an adenine nuclear thallium or thymidine residue, and X9 can bind to complementary nucleotides on the acceptor nucleic acid sequence, two nucleotides upstream from the cleavage position. Confirm that the functional part includes an upstream flanking region and a downstream flanking region. The upstream flanking region is immediately after the catalytic functional site 5 ', and the downstream flanking region is immediately adjacent to the catalytic functional site 3', as described in the catalytic DNA molecule above. , Which can cleave ribonucleotides. — 87851 200413528 In some specific examples of the catalytic DNA in this aspect of the present invention, X2 is a thymine residue, X3 is a cytosine residue, X4 is a thymidine residue, and X5 is a thymidine residue, X6 is a thymidine residue, X7 is an adenine nuclear crest residue, and X8 is an adenine nuclear crest residue. In a specific embodiment of the preferred embodiment of the catalytic DNA in this aspect of the present invention, the loop contains V-TCGTCTTAGACA3 '(SEQ ID NO: 30). In another aspect, the present invention provides a receptor nucleic acid sequence, which includes a non-naturally occurring ribonucleic acid, immediately upstream of a cleavage site, and is flanked by a complementary sequence that can bind to a binding region, also referred to herein as a catalyst 4 flanking regions of sex DNA molecules. In some specific examples, the non-naturally occurring ribonucleic acid of the subject nucleic acid sequence is 2 ', 5' ribonucleic acid, preferably adenine nucleotide or guanine nucleotide residue, and most preferably Is a guanine nucleotide residue. In other specific examples, the non-naturally occurring ribonucleotide of the acceptor nucleic acid sequence is the L-enantiomer of a 3 ', 5'-ribonucleotide, preferably an adenine riboic acid residue. In another aspect, the present invention proposes a non-naturally generated single-stranded nucleic acid substrate as described above, which includes a pair of a first flag and a second flag, which are separated by a cutting position in 4 phases, The mutual activity standard. Flags can be attached directly or indirectly to a single stranded nucleic acid substrate. In some specific examples, the first flag is a fluorescent part and the second flag is a coolant. When both the fluorescent part and the coolant are adhered to a single-stranded nucleic acid substrate, the latter can cool the fluorescent part. Serving. In another aspect, the present invention proposes a method for detecting a target nucleic acid sequence, wherein a catalytic nucleic acid molecule and a acceptor nucleic acid sequence of the present invention are used. The method is related to the method used for quantitative PCR and is called nDzyNA-PCR ''. Therefore, the method includes blending the following components in the following expansion buffer solution:-10-S7851 200413528 0 Phosphonic acid sample ii) Polymerase

Hl) A single-stranded nucleic acid sequence that is not naturally generated by the substrate, and includes a pair of mutually active flags consisting of a first flag and a second flag (which can be directly or indirectly attached to oligonucleotides), of which the first flag Separated from the second flag by the above-mentioned non-naturally generated ribonucleic acid cleavage site, · W) —a forward primer that can be related to the first strand of the target nucleic acid sequence], part, mouth , And direct the synthesis of the target nucleic acid sequence in the nucleic acid sequence; and &quot; &quot; ν)-an opposite primer, including a region that can be combined with the target nucleic acid sequence, the second leg of the 3 foot content, In addition, the target nucleic acid sequence can be used to synthesize polynucleic acid in the reverse direction, and includes the complement of a catalytic single-stranded DNA molecule, wherein the catalytic single-stranded DNA molecule can cleave the acceptor nucleic acid sequence at the cutting position. &lt; Post-manipulation methods include incubating the incorporated components under expansion conditions to expand the target nucleic acid sequence. This results in the synthesis of catalytic single-stranded DNA molecules. The resulting synthesis catalyzes a single-stranded DNA molecule, and then cuts the acceptor nucleic acid sequence, which is the interaction between the first flag and the second flag. Then the first flag is detected, so that the target nucleic acid sequence is detected. In the exemplary embodiment of the method, the non-naturally occurring ribonucleoside d-position includes 2 ', 5' linked residues or L · enantiomer residues, such as the above-mentioned subject matter nucleic acid sequences of the present invention. Furthermore, the catalytic single-stranded molecule is preferably one of the above-mentioned catalytic single-stranded DNA molecules. The present invention also includes the use of direct development methods to generate nucleic acid molecules with predetermined catalytic activity 87851 -11- 200413528 to cleave single-stranded nucleosides including non-naturally occurring ribonucleotides. Therefore, in another aspect, the present invention A method for identifying catalytic DNA molecules with position-specific endonuclease activity is proposed, which is specific to the position of the non-naturally occurring ribonucleic core. The method includes constructing a double-stranded nucleic acid molecular library, which includes a non-naturally occurring ribonucleic acid cleavage site, and which includes a first sequence of nucleotide regions that may interact with the cleavage site S region. &lt; After capturing one strand of the double-stranded nucleic acid molecular library, a captured single-strand nuclear I knife library is provided. Next, the captured single-stranded nucleic acid knife library is grown under cleavage conditions so as to be able to cut at the cleavage position and release the cut nucleic acid molecules. Thereafter, the cleaved nucleic acid molecule is isolated, so that a catalytic DNA molecule having a position-specific endonuclease activity is identified, which is specific for a non-naturally occurring ribonucleotide cleavage position. 2. In the illustrated specific example, the method further includes expanding the cut nucleic acid knife and repeating the above-mentioned capture, cutting and separation steps between 50 and 50 times, L reed soil between 20 / person. The selectively amplified cleaved nucleic acid molecule can be arbitrarily large and sufficient to form a cleaved and cleaved nucleic acid molecule. The mutated and cleaved nucleic acid molecule is extended to capture, cleave, and isolate steps between 1 and 20 times. In the specific examples described, as shown in the following example, the method of cutting the diced pieces can be divided into two groups, so that the cleavage reaction must be more efficient so that the cleavage of the nucleic acid can be performed in the subsequent cutting step. occur. In the specific example, the 'non-naturally occurring ribonucleic acid cleavage position is 2,3 _link &lt; guanine nucleotide ribonucleotide cleavage position or L- adenine nucleotide ribonucleotide cleavage position . Ben Gengming also produced kits, including catalytic DNA molecules and / or receptors 87851 -12-200413528, which have non-naturally occurring ribonucleotide. In another embodiment, the set includes primers, polymerases, and other reagents useful in the methods of the present invention. [Embodiment] The present invention provides a catalytic single-stranded DNA molecule, which has position-specific endonuclease activity, and is specific for the cleavage position in the subject nucleic acid sequence, wherein the cleavage position includes an unnaturally generated single-stranded DNA molecule. RNA, or a combination thereof. Catalytic DNA molecules include more than one circular band region and more than one binding region, wherein the binding regions can bind the complementary sequence of the acceptor nucleic acid sequence. In a preferred embodiment, the non-naturally occurring single-stranded nucleic acid includes 2 ', 5 · -linked residues, preferably 2', 5'-linked adenine nucleotides, or most preferably 2 ', 5 '-Linked guanine nucleotide ribonucleotide residues, or L-enantiomeric residues, preferably L-enantiomer 3', 5 ^ -linked adenine ribonucleotide ribonucleotide residues base. As used herein, the term `` catalytic DNA molecule '' is used to describe a nucleic acid containing DNA, which acts as an enzyme. In this disclosure, "deoxyribonuclease" includes endonucleases and endonucleases. Enzymes, however, deoxyribonuclease has endonuclease activity. Other terms that can be used interchangeably with deoxyribonuclease are "enzymatic DNA molecules", "nDNA enzyme" or "deoxyribonuclease π", which should be understood to include Its enzyme-active part, whether synthetic or derived from an organism or other source. The catalytic DNA molecule of the present invention generally includes a DNA molecule that is targeted to a specific oligonucleotide or is complementary to a substrate in a substrate-binding region; this molecule also has enzyme activity and has specific cleavage oligonucleotide Qualitative activity. Enzymatic DN A molecules can cleave oligonucleotide substrates between molecules. This complementary function allows the enzymatic DNA molecule to fully hybridize with the receptor oligonucleotide, and intermolecular cleavage of the receptor -13-87851 200413528 can occur. Although 100% complementarity is preferred, &lt; complementarity in the range of 75-100%, such as 90%, 95%, and 99% complementarity are also useful and are encompassed by the present invention. The enzymatic DNA molecule of the present invention may be further described as having nuclease or ribonuclease activity. These terms are used interchangeably herein. As used herein, "enzymatic nucleic acid" includes an enzyme RNA or DNA molecule, an enzyme RNA-DNA polymer, and an enzyme active moiety or derivative thereof, although an enzyme DNA molecule is a type of the enzyme-active molecule of the present invention. In one aspect, the present invention proposes a catalytic single-stranded DNA molecule that can cleave the mitochondrial M sequence, which includes 2,5, -linked ribonucleotides, such as 2 ', 5'-linked adenine Nucleic acid or guanine ribonucleotide ribonucleotide residues, as shown in Figure 14A. The catalytic DNA molecule of this aspect of the present invention has a catalytic function site and a confirmation function site (15, 25). The sexual function part includes a downstream region, which includes the sequence S'-XiX ^ CTCGGAGXy '(SEQ ID NO: 28), which can form an annulus; a central stem-stalk region is immediately adjacent to 5 of the downstream annulus, and has The sequence, and an upstream region immediately adjacent to the central stem region, can form a loop with the sequence 5'-GGGA-3 '. The loop formed in the catalytic DNA molecule in this aspect of the present invention is internally expanded. Circumferential zone. &Amp; is the required cell X2 is a nucleoside residue, X2 is a cytosine nucleoside or thymosine nucleoside residue 'and &amp; can be bound to the nucleic acid sequence of the complementary nuclear acid, two nucleotides upstream of the cleavage position. Z4 may Binds to and / or forms a oscillating pair on the acceptor nucleic acid sequence, immediately downstream of the cleavage site. In some specific examples, Z2 is a cytosine nucleoside residue, and 4 is a vein 87851- 14- 200413528 residues. The confirmatory function includes an upstream flanking region (25) and a downstream flanking region (15). The upstream flanking region (25) is immediately adjacent to the upstream loop zone: and the downstream side The junction region (5) is immediately downstream of 3. In various embodiments of the present invention, the side junction region has various length changes. Therefore, the example of the side junction region may include from a single nuclear to 75 nuclear ^ : Acid. 'However, it should be understood that the lateral region of about 3_25 nuclear #acid length, preferably about 3_15 nuclear acids, and preferably about 3_ιο nuclear acids are characteristic. In various specific examples In the flanking region, individual nuclear fenamic acids can form complementary test pairs with the nuclear acid of the acceptor molecule; In the conventional examples, non-standard paired interactions can be formed. Complementary and non-standard pairings are also within the scope of the specific examples disclosed in the present invention. As described in some specific examples, the present invention proposes a catalyst Single-stranded cricket molecule that can cleave the acceptor nucleic acid sequence, which includes 2,5, linked ribonucleosides, preferably 2,5, _ linked adenine or ornithine Residues, as shown in Figure 14A. However, the methods disclosed herein related to the use of direct expansion can be used to catalyze more catalytically single-stranded DNA molecules that are not naturally occurring nucleic acids. For example, catalytic single-stranded molecules can Cutting such as: 2'_deoxy_2, _amino group core #acid, 2, _deoxy. Wide-band nuclear material, nuclear thioscale acid, nuclear tritium, · 15,) phosphoramidate, arabinose #, And various RNA analogs containing Jing Xiu's test base. In another embodiment, the catalytic molecules of the present invention may further include a third lateral region. In some specific examples of the present invention, the catalytic DNA molecule may further include _ $ 夕 ^ 祜 or more variable or, the "spacer" region is between the flanking regions. 87851 -15 · 200413528 In the present invention In some specific examples of this aspect, the downstream loop has a sequence of 5'-CCACTCGGAG-3f (SEQ ID NO: 22). In some specific examples, the upstream loop has a sequence of 5'-YGGGA-3 ', Wherein Y is 0 to 5 nucleotides. For example, Y may include the sequence 5'-TTA-3 ', for example, Y may be 5'-GTTTA-3, (SEQ ID NO: 19), 5, -GCTTA- 3, (SEQ ID NO: 20), 5f-GTTA-3, (SEQ ID NO: 21). In some specific examples, z4 is a guanidine residue, Xi is a cytosine nuclear pyrene residue, and X2 is Cytosine nuclear pyrene residues, and X3 is a guanidine residue. In another preferred embodiment as illustrated in Figures 2a, 2b and 9a ^, the catalytic DNA molecule includes SEQ ID NO: 1, SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. SEQ ID NO: 1-8 does not include the upstream flanking region, so this flora A fixed sequence region that is not allowed to be mutated during expansion, as described in the example. In a particular embodiment, the catalytic DNA includes SEQ ID NO: 10. In another aspect illustrated in Figure 14B, the present invention A catalytic single φ strand DNA molecule is proposed, which can cleave the acceptor nucleic acid sequence and column on the L-ribonucleic acid residue, such as 1,5'-linked L-adenine nucleotide ribonucleic acid residue In this regard, a catalytic DNA molecule includes a confirmatory functional site and a catalytic functional site capable of forming a loop band, which includes the nucleic acid sequence 5t-X1X2X3GX4CX5X6X7GACX8X9-3 '(SEQ ID NO: 29). The loop band is an internal expansion X! Can bind a complementary nucleotide on the acceptor nucleic acid sequence, which is located immediately downstream of the cleavage site on the acceptor nucleic acid sequence, 乂 21 is the thymic nucleus or guanidine residue, and X3 is the cell Nuclear pyrene or guanidine residues, X4 is the cytosine 87851 -16- 200413528 pyrimidine or thymidine residue, X5 is the cytosine or thymidine residue, and χ6 is the cytosine or thymidine core Glycoside residue, x7 is adenosine or guanidine residue, x8 is adenosine or thorax Nucleic acid residues, and X9 is bound to a complementary nucleotide on the acceptor nucleic acid sequence, two nucleotides upstream from the cleavage position. Confirm that the functional site includes an upstream flanking region and a downstream flanking region, upstream The flanking region is immediately after 5 'of the catalytic function site, and the downstream flanking region is immediately after 3' of the catalytic function site, as described above for the catalytic 0 DNA molecule that can cleave 2 ', 3' ribonucleotides. In the preferred embodiment of the present invention = catalytic DNA in this aspect, X2 is a thymine residue, X3 is a cytosine residue, X4 is a thymine residue, X5 is a thymus nucleoside residue, X6 Is a thymic nucleus residue, X7 is an adenosine residue, and X8 is an adenosine residue. In a specific example of a preferred specific embodiment of the catalytic DNA in this aspect of the invention, the loop contains 5TCGTCTTAGACA 3 '(SEQ ID NO: 30) ° As illustrated in Figures 2C and 9B, in some preferred specific examples In the present invention, the catalytic DNA molecules of this aspect include: SEQ ID NO: 11, SEQ ID NO.12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID N O "6, SEQ ID NO: 17, or SEQ ID NO: 18. SEQ ID NOs: 11-17 do not include the upstream flanking region because this region is in a fixed sequence region and it must not be mutated during expansion, as described in the examples. The endonuclease activity of the catalytic DNA molecule of the present invention generally involves the hydrolytic cleavage of a phosphate bond at the cleavage site. In some specific examples of the present invention, the acceptor nucleic acid sequence and the catalytic DNA molecule are part of the same DNA molecule ~ 87851 -17- 200413528. The endonuclease activity of catalytic DNA molecules is usually enhanced by the presence of certain metals, especially divalent cations, as shown in Figure 12. In some specific examples, the catalytic DNA molecule can catalyze the cleavage of 2 ', 5' ribonucleic acid, and the activity of the catalytic DNA molecule can be enhanced by the presence of Mg2 +, Sr2 +, Ca2 +, or Ba2 +, especially Mg2 +. In other specific examples, the catalytic DNA molecule can catalyze the cleavage of L-ribonucleic acid, and the activity of the catalytic DNA molecule can be enhanced by the presence of Mn2 +, Mg2 +, Sr2 +, Ca2 +, Pb2 + or Zn2 +, especially Mn2 + And Mg2 +. The catalytic DNA molecule and the acceptor nucleic acid sequence of the present invention may include a nucleotide-analog. A "π nucleotide analog" generally refers to a purine or pyrimidine nucleotide that is structurally different from A, T, G, C, or U, but has sufficient similarity to replace a normal nucleotide in a nucleic acid molecule. As used in this article, `` nucleotide analogs π include different bases, different or unusual sugars (ie, non-'' normal '' ribofuranosyl sugars), such as L-ribose instead of D-ribose, or different bases and A combination of different or unusual sugars. φ In various specific examples, the catalytic DNA molecules of the present invention can be mixed with one or more modifications or mutations, including the addition, deletion, and substitution of the special catalytic DNA molecular sequence disclosed herein, which is based on the use of technology Well-known methods were introduced. In various specific examples, this mutation or modification can be produced by a method which generates any or specific mutation or modification. These mutations, for example, can change the length of loop bands, space subregions or confirmation sequences (or functional sites), or change the nucleotide sequence. One or more mutations in one catalytically-active enzyme DNA molecule can be combined with mutations in the second catalytically-active enzyme DNA molecule 87851 -18- 200413528 to produce novel enzyme properties containing two molecular mutations DNA molecule. The catalytic DNA molecule of the present invention may have various lengths and folding patterns depending on the type and function of the molecule, as appropriate. For example, catalytic DNA molecules can be about 15 to about 400 or more nucleotides in length, but typically no more than about 250 nucleotides, to avoid limiting the therapeutic use of the molecule because it is too large or difficult to handle. In certain embodiments, the catalytic DNA molecules of the present invention include no more than about 100 nucleotides. In still other specific examples, the catalytic DNA molecule of the present invention is about 20-75 nucleotides long, preferably about 20-65 nucleoside φ acids. Other preferred catalytic DNA molecules are about 10-50 nucleotides in length. In other applications, the configuration of the catalytic DNA molecule can be similar to the "hammerhead" ribozyme. This catalytic DNA molecule usually does not exceed about 75-100 nucleotides, or about 20-50 It should also be understood that the catalytic DNA molecule of the present invention may include the enzyme active portion of the catalytic DNA molecule, or may include one or more mutated catalytic DNA molecules, and if one or more of them can form The base pair sequence or space does not exist or has been modified, as long as the deletion, addition or modification will not detrimentally damage the ability of spring molecules to behave like enzymes. "Oligonucleotide or polynucleotide" Usually refers to a polymer of single or double stranded nucleotides. As used herein, "oligonucleotide π and its grammatical equivalents include the full range of nucleic acids. Oligonucleotides generally refer to nucleic acid molecules containing ribonucleotides or deoxyribonucleic acid linear strands. The exact size depends on a number of factors, which in turn depends on the end use conditions, as is well known in the art. The catalytic nucleic acid molecules of the present invention also include those having different confirmed positions or functional sites. In various specific examples, these altered confirmatory functional sites can provide unique sequence specificity on fermented nucleic acid molecules, including this confirmatory functional site. The exact bases present in the confirming functional site determine the sequence of bases where cleavage can occur. The cleavage of the host nucleic acid occurs within the identified functional site. This cleavage can leave 2 ', 3' or 2 ', 3'-cyclic phosphate groups on the cleavage sequence of the substrate, and a 5' hydroxyl group on the nucleic acid, which was originally directly adjacent to the original substrate. 3 'of the cleavage sequence. The cleavage can then be directed to the preferred location, which is a change in the test substrate present in the receptor-binding region of the catalytic DNA molecule (also known as the confirmation sequence (internal guide sequence)). See Murphy et al., Proc. Natl. Acad · Sci. USA 86 · 9218-9222 (1989).瘪 Furthermore, the addition of polyamines may be useful to facilitate the identification and binding of DNA molecules and their substrates. Examples of useful polyamines include spermidine, putrescine or spermine. In a specific embodiment, spermidine is effective at a concentration of about 1 mM, and a concentration range of about 0.1 mM to about 10 mM is also useful. In various specific examples, the catalytic DNA molecules of the present invention have enhanced or more perfect ability to cut nucleic acid substrates, preferably RNA substrates. As understood by the artisan, the rate of enzyme-catalyzed reaction varies depending on the substrate and enzyme concentration, and generally, it can be flattened &lt; at high substrate or enzyme concentrations. Taking this effect into account, the kinetics of the enzyme-catalyzed reaction can be described in the terms that define the reaction below. The ability of the catalytic DNA molecules of the present invention to enhance or improve the ability to cut RNA substrates can be determined in the cleavage reaction, wherein the amount of labeled RNA substrates is changed in the presence of the catalytic DNA molecules. The ability to cleave a substrate is usually defined by the catalytic rate (Keat) divided by the Michaelis constant (Km).

The Kcat symbol represents the maximum rate of enzyme response as the substrate approaches saturation. KM--20- 87851 200413528 represents the mass concentration when the reaction rate is half of the maximum value. For example, the κM and Kcat values of the present invention can be determined experimentally, where the substrate concentration [S] exceeds the concentration [E] of the catalytic DNA molecule. The initial reaction rate (v0) in the mass concentration range is estimated from the initial linear phase, usually below 5% before the reaction. The data points are combined with the theoretical straight line by the least square method, and the equation is used: ν = -ΚΜ (ν〇 / [3]) + νπ ^ χ. Therefore, Keat and KM can be determined by the initial reaction rate, v0, and the mass concentration [S]. In various specific examples, the catalytic DNA molecules of the present invention have enhanced or improved ability to cut nucleic acid substrates with non-naturally occurring ribonucleotides. In many specific examples, the catalytic DNA molecules are cleaved. RNA-enhanced or more refined ability of a substance, showing at least 50-fold regioselectivity or enantiomer. Typically, catalytic DNA molecules exhibit a Km value of less than about 1 μM, or in some specific examples, less than about 100 nM. The skilled artisan should understand that the enhanced or improved ability of the catalytic DNA molecule to cleave the nucleic acid substrate can vary depending on the selection limitations applied in the in-tube expansion method of the present invention. · The following examples further describe various methods of altering the present invention's DNAzymes and others. Enzymatic DNA molecules and nucleases. The present invention also proposes a composition containing one or more types or groups of catalytic DNA molecules of the present invention. Different types or groups can identify and cleave different nucleotide sequences. The composition may further contain a ribose-containing substrate. The composition according to the present invention may further be lead ions, magnesium ions, or other divalent or monovalent cations, as discussed herein. Preferably, the content of the catalytic DNA molecule is from about 0.05 μM to about 2 μM. For Code 87851 -21-200413528, the content of catalytic DNA molecules is from about 1: 5 to about 1:50. More typically, the catalytic DNA molecule is present in the composition at a concentration of about 0.1 μM to about 1 μM. Furthermore, the composition may contain a catalytic DNA molecule at a concentration of about 0.1 μM to about 0.5 μM. The content of the substrate in the composition is from about 0.5 μM to about 1000 μM. As mentioned previously, magnesium ions, lead ions, or other suitable monovalent or divalent cations can be present in the composition in an amount of about 1-100 mM. In comparison, the content of the selected ion in the composition is about 2 mM to about 50 mM, and about 5 mM is particularly preferred. The artisan understands that the concentration of 100 mM ion is only limited by the solubility limit of its source (such as magnesium) in aqueous solution, and it is expected that the catalytic DNA molecule can exist in the active configuration in the same composition. The optimum cation concentration to be included in the nucleic acid cleavage conditions can be easily determined by the content of the single-stranded nucleic acid cleaved at a specific cation concentration. The artisan should understand that the optimal concentration will vary depending on the particular catalytic DNA molecule used. The nucleotide sequence of the functional site is confirmed by changing the catalytic DNA molecule, and the disclosed method can cleave on any nucleotide sequence. In this way, single-stranded nucleic acids can still be cleaved without the restriction enzyme position at the selected position. The effective dose of a catalytic DNA molecule is the amount necessary to cleave a predetermined base sequence in a single-stranded nucleic acid. Typically, the content of catalytic DNA molecules is a molar ratio of DNA molecules to the cleavage site of the substrate from 1 to 20. This ratio can be varied depending on the length of the treatment and the efficiency of the particular catalytic DNA molecule under the particular nucleic acid cleavage conditions applied. Therefore, in a specific example, processing usually involves mixing RNA-containing substrates and enzymes in an aqueous solution to form a cleavage admixture, and then maintaining the adduct such as -22- 87851 200413528 under conditions of RNA cleavage. For a sufficient period of time, the catalytic DNA molecule can cleave the RNA duplex on any predetermined nucleotide sequence in the ruler. Ion sources are also provided in various embodiments, such as monovalent or divalent cations, or both. In a specific example of the invention, the time required for a catalytic DNA molecule to cleave a single-stranded nucleic acid has been determined. The time ranges from about 1 minute to about 24 hours, and depends on the concentration of the reactants and the reaction temperature. Generally, this time is from about 10 minutes to about 2 hours' so that the animal DNA molecule can cleave a single-stranded nucleic acid on any predetermined nucleotide sequence. The invention further includes nucleic acid cleavage conditions, including a pH value from about pH 60 to about pH 9.0. In a specific example, the pH is from about pH 6.5 to pH 8.0. In another specific example, the pH is as physiological as possible, that is, pIi is about 7.0--7, and pH 7.5 is the most typical. Those skilled in the art should understand that the method of the present invention can be operated at a wide range of pH values, as long as the pH value used for nucleic acid cleavage allows the catalytic DNA molecules to be maintained in the active configuration. Catalytic DNA molecules in an active configuration can be easily measured for their ability to cleave single-stranded nucleic acids on a predetermined nucleotide sequence. In various specific examples, nucleic acid cleavage conditions also include various temperature ranges. As mentioned earlier, the temperature range that meets physiological conditions is particularly preferred, but the temperature range that meets individual applications is also included here. In a specific example, the temperature range is from about 15 ° C to about 60 ° C. In another variation, the nucleic acid cutting conditions include a temperature from about 30 ° C to about 56 ° C. In yet another variation, the nucleic acid cleavage conditions include from about 35 ° C to about 50 ° C. In still another preferred embodiment, the nucleic acid cleavage conditions include a temperature range from about 37 ° C to about 42 ° C. 87851 -23-200413528 The temperature range that meets nucleic acid cleavage conditions is limited only by the desired cleavage rate and the specific catalytic properties at special temperatures. The invention also describes a performance vector, which includes a nucleic acid fragment encoding the catalytic test molecule of the present invention and located in the vector, in a way that allows the catalytic DNA molecule to be in the target cell (such as a plant or animal cell). As described above, the present invention "catalytic DNA molecule can cut the acceptor nucleic acid sequence (Figures 14A and 14B (upper strand)), which includes non-naturally generated riboflavin acid immediately upstream of the cutting position (arrow), and its side A complementary sequence that can be bound to a binding region is also referred to herein as a flanking region and is a catalytic DNA molecule. The acceptor nucleic acid sequence itself is another aspect of the invention. In certain preferred embodiments, the non-naturally occurring ribonucleic acid of the subject nucleic acid sequence is 2, 5, ribonucleotides, such as adenine nucleotides or guanine ribonate residues. In another specific example, the non-naturally occurring ribonucleoside acid of the receptor nucleic acid sequence is the L-enantiomer of 3,5, ribonucleic acid, preferably a guanine ribonate residue. In another aspect, the present invention proposes a non-naturally generated single-stranded nucleic acid substrate as discussed above, which includes an interaction flag pair consisting of a first flag and a second flag. Separated. Flags are either directly or indirectly attached to single-stranded nucleic acid substrates. As discussed above, the cleavage position includes residues of 2, 5, 5, or l-enantiomers. The first flag may be the fluorescent part, and the second flag is the quenching agent. When the fluorescent part and the quenching agent are both adhered to the single-stranded nucleic acid substrate, the cooling agent may cool the fluorescent part. Serving. The acceptor nucleic acid sequence and the catalytic DNA molecule of the present invention do not substantially correspond to the share. It is particularly useful as a biochemical tool in cleavage informant molecules, such as the double-labeled acceptor nucleic acid sequence 87851 -24- 200413528 containing the above-mentioned non-natural ribonucleotides. This informant is not cleaved by the biological nuclease. Therefore, one application of the catalytic nucleic acid molecule and the acceptor nucleic acid sequence of the present invention is a method related to quantitative PCR, which is called "DzyNA-PCR" (pictured in Figure 16; see Todd, A. V., et. al., Clin. Chem. 46, 625-630 (2000), which is incorporated by reference in its entirety; see Applegate et al. Clin. Chem., 48; 133 99-1488 (2002), which has been incorporated by reference in its entirety. ) This method uses a DNA enzyme that can cleave RNA to cleave the reporter oligonucleotide, which contains a fluorescent marker and a coolant on both sides of the cleaving position. The DNA enzyme sequence is encoded by a complementary sequence that attaches φ to the 51 end of one of the two PCR primers. When the PCR amplification effect is performed, it can generate a set of functional DNA enzymes, which can cleave the reporter molecules, so that the fluorescent marker and the coolant are separated, and a signal with fluorescence is generated. In the existing method, because the informant contains natural ribonucleic acid, it is easily cut by biological ribonuclease. However, this is not the case when the method utilizes a catalytic DNA molecule that can cleave non-natural ribonucleic acid within the informer molecule. There are no known biological nucleases that can cleave 2 ', 5'-linked guanine ribonucleic acid or L-ribonucleic acid. φ Therefore, another aspect of the present invention is to propose a method which comprises admixing the following components in an expansion buffer solution: i) a nuclear fei sample, ii) a polymerase; m) a single-stranded nucleic acid that is not naturally produced by the substrate A sequence containing a pair of markers consisting of a first marker and a second marker and directly or indirectly attached to the interactive activity of the oligonucleotide, wherein the first marker and the second marker are unnatural The generated ribonucleic acid is cleaved by the cleavage position, as discussed above; -25- 87851 200413528 Π〇 a forward primer that can bind to the first 3, part of the target nucleic acid, and instruct the target nucleic acid sequence Polynucleotide synthesis in the forward direction; and V)-a reverse primer, including a region that can bind to the second 3, part of the target nucleic acid sequence, and can direct the target nucleic acid sequence to polymerize in the reverse direction The synthesis of nuclear I and fe and includes the complementary single-stranded Dn a according to the present invention, in which the catalytic single-stranded DNA molecule can cleave the nucleic acid sequence at the cutting position. Subsequent methods include incubating the incorporated components under expansion conditions to expand the target nucleic acid sequence. This enables the synthesis of catalytic single-stranded DNA molecules. The resulting catalytic single-stranded DNA molecule can cleave the acceptor nucleic acid sequence again, thus releasing the interaction of the first and second flags. The first flag can be detected to detect the target nucleic acid sequence. In certain specific examples of the method, the non-naturally occurring ribonucleotide cleavage positions include 2 ', 5' linked residues or L-enantiomeric residues, as described above for the subject nucleic acid sequences of the invention. Furthermore, the catalytic single-stranded DNA molecule is usually one of the above-mentioned preferred catalytic single-stranded DNA molecules. For example, when the ribonucleic acid cleavage position is 2 ', 51-linked adenopharyngeal nucleophilic acid or ornithine ribonucleotide ribonucleic acid ribonucleic acid residue', the catalytic DNA molecule may include a confirmatory functional site and A catalytic functional site can form a loop containing the nucleic acid sequence S'-XiXAsGXWXsXsXAACXsXrS, (SEQ ID NO: 29). The amplification step used for the method of the present invention for detecting the target nucleic acid sequence is usually a polymerase chain reaction (PCR), as is well known in the art, and is described in, for example, 口 .8.? &Amp; 1:.] ^ 〇3.4 Nos. 683, 195, 4, 683, 195, and 4,800, 159 are listed in their entirety for reference. When PCR is used as the expansion method, the polymerase is aquatic habitat heat -26-87851 200413528 bacteria (Thermus aquaticus (Taq)) DNA polymerase. A nucleic acid sample can be virtually any nucleic acid. In some specific examples, the nucleic acid sample can be isolated from a natural source. For example, the nucleic acid can be genomic DNA or RNA, such as mRNA (see Applegate et al. (2002)). In the detection of the target nucleic acid sequence, other amplification methods other than PCR can also be applied in the present invention. The limitation is that the nucleic acid molecules used in the amplification method must have complementary primers and catalytic DNA molecules. In one method, the PCR method is performed in an automated manner, in which enzymes that are stable to heat are used. In this method, the reaction mixture undergoes cyclic steps such as denaturation, primer cooling, and genseng, so that the nucleic acid substrate can be cleaved in the polymerase synthesizing catalytic DNA molecule. Commercial equipment that can perform this method is available from Perkin-Elmer Cetus Instruments and is designed to use thermostable enzymes. In detecting the target nucleic acid, the method of incorporating the flag and target pair of the acceptor nucleic acid sequence of the present invention can be found in US Pat No. 6,214,979, which is incorporated herein by reference in its entirety. Various flags suitable for use in the method of the present invention, as well as methods in which the acceptor nucleic acid sequence is contained, are well known in the art. The flags include the following but are not limited to this: Oxidase), and enzyme substrates, radioactive atoms, fluorescent dyes, chromophores, chemiluminescence flags, chemiluminescence flags, such as Origin ™ (Igen), ligands with special binding pairs, or other Any sign that can interact to enhance, change, or eliminate the signal. The "flag" as used herein refers to any atom or molecule that can provide a measurable and preferably quantifiable signal and that can be attached to a nucleic acid or protein. -27- 87851 200413528 t 幡 can provide measurable signals through fluorescence, radioactivity, colorimetry, gravimeter, X-ray diffraction or absorption, magnetization, and enzyme activity. When constructing the labeled probe of the present invention, the fluorophores that can be used as flags include rhodamine and derivatives such as Texas Red, fluorescein and derivatives such as 5.bromomethylfluorescence. , Lucifer Yellow, IAEDANS, 7-Me2, N_coumarin-4_acetate, 7-OH-4_CH3-coumarin-7 · acetic acid '7-NH2-4CH3-coumarin-3_acetic acid Ester (AMCA), monochromometer, monotrisulfonate, such as Cascade Blue, and monobromo dimethyl-ambanaide. Generally speaking, it is better to use a fluorescent group with wide stokes shifts. This is done by using a filter-based fluorometer instead of a monochromator, and to increase the detection efficiency. As mentioned above, two cross-active flags are usually used on a single nucleic acid sequence. However, it is necessary to consider that the flags must be kept at an appropriate distance on the nucleic acid molecule so that the flags can be separated during the hydrolysis of the nucleic acid sequence. . In a preferred embodiment, the acceptor nucleic acid sequence can be labeled with a fluorophore and a refrigerant. When normal positive nucleic acid sequences are used, the fluorescence of the fluorophore can be repaired by the coolant. In this method, the duplex nucleic acid sequence is cut between the fluorophore and the coolant. Where all show their fluorescence. The interaction of flags, such as the glory of disgrace τ 7 and the energy transfer between the first and second flags (such as fluorophores and refrigerants) and the emission spectrum of this fluorescent group and the absorption spectrum of refrigerants Necessary ® The preferred combination of this aspect of the invention is the fluorescer-rhodamine 590 plus the quencher-crystal violet. In order to detect hydrolyzed labeled probes, such as polarized light can be used. This technology is based on molecular _biing, which can distinguish between large and _ 87851 -28-200413528 small molecules. The large molecules (such as intact labeled probes) are slower in solution than the smaller molecules. Once the fluorescent portion is connected to the key molecule (such as the end of the labeled probe), this fluorescent portion can be inverted according to the molecule as Basically determined (and differentiated), thus distinguishing between intact and hydrolyzed probes. In the method of the present invention, if a labeled nucleic acid sequence is used in the PCR for the substrate, the flag can be directly detected in the PCR, or detected after the PCR. When one of the marker pairs is a fluorescent marker, you can use various characterization equipment designed for fluorescent marker analysis, such as the ABI Gene Anglyzer to analyze attomole quantities of DNA with spring fluorescent groups, such as ROX (6 leptyl-X-rhodamine) 1 rhodamine-NHS, TAMRA (5/6 • carboxytetramethylrhodamine NHS), and FAM (5 ′ • carboxyfluorescein NHS). These compounds can be attached to the probe by an amidine bond via a 5'-alkylamine of the probe. Other useful fluorophores include CNHS (7-amino-4-methyl-coumarin-3-acetic acid, succinimide), which can also be attached via an amidine bond. Using commercially available amino phosphite reagents, we can generate appropriately protected amino phosphites containing acceptor nucleic acid sequences containing functional groups (such as thiols or primary amines) at the 5 'or 3' end, and can utilize The above-mentioned strategy labeling, such as PCR j Protocols: A Guide to Methods and Applications (Innis et al.? Eds. Academic Press, Inc., 1990) 〇 The present invention also includes a method for generating a nucleic acid molecule using a direct expansion method, It has a predetermined catalytic activity to cleave single-stranded nucleic acids, including non-naturally occurring ribonucleic acid. An overview of the method for direct in-tube expansion of an enzymatic DNA molecule can be found in Example 1 of 1 ^ &amp; 1 &gt; 6,3265174, which is incorporated herein by reference in its entirety. 87851 -29- 200413528 In-tube selection and in-tube expansion techniques allow new catalysts to be separated even without prior techniques of their composition or structure. This method has been used to isolate RNA enzymes with novel catalytic properties. For example, ribozymes that are feasible for autolytic cleavage of heterocations have been derived from any tRNAPhe subpopulation (Pan and Uhlebeck, Biochemistry 31: 3887-3895 (1992)). A ribozyme variant of group I has been isolated that can cut DNA (Bedudry and Joyce, Science 257: 635_641 (1992)) or that it has different metal dependencies (Lehman and Joyce, Nature 361: 182-185 (1993)) . From the collection of arbitrary RN A · sequences, molecules that can catalyze polymerase reactions can be obtained (Bartel and Szostak / Science 261: 1411-1418 (1993)) ° Therefore, in another aspect, the present invention proposes to identify catalytic properties The method of DNA molecules has position-specific endonuclease activity and is specific for non-naturally occurring ribonucleic acid cleavage sites. The method includes constructing a double-stranded nucleic acid molecular library, which includes non-naturally occurring ribonucleotide cleavage sites, and includes a region of any sequence of nucleotides that can potentially interact with the cleavage site region. Next, capturing one strand of a double-stranded nucleic acid molecular library may call for the provision of a single-stranded nucleic acid molecular library captured. Next, the captured single-stranded nucleic acid; molecular library is incubated under cleavage conditions so that it can be cut at the cleavage position and the cleavage nucleic acid molecules are released. Isolation of the cleaved nucleic acid molecules can then identify catalytic DNA molecules with position-specific endonuclease activity that are specific to the cleavage position of the non-naturally occurring ribonucleic acid. In some specific examples, the method further comprises expanding the cleaved nucleic acid molecule and repeating the capture, cleavage and separation steps 1 to 50 times, usually 1 to 20 times. The selectively cleaved nucleic acid molecule can be mutated arbitrarily to form mutation 87851-30 · 200413528 疋 cleaved nucleic acid molecule. The mutated nucleic acid molecule expands this capture, and the cleavage and separation steps are about 1 to 20 times. In some specific embodiments, the non-naturally occurring ribonucleotide cleavage position is a 2 ', 5'-linked guanine nucleotide ribonucleotide cleavage position, or a glandular ribonucleoside ribonucleotide cleavage position. The example illustrates a method of constructing a double-stranded nucleic acid molecular library, which includes a non-naturally occurring ribonucleotide cleavage site, and a nucleotide region of any sequence therein, which can potentially interact with the cleavage site region. In this non-limiting example, the double-stranded nucleic acid molecules in the library can be synthesized by chemically synthesizing overlapping oligonucleotide pairs to produce a first oligonucleotide in the pair that is synthesized at a non-naturally occurring ribonucleotide cleavage position, and a second Oligonucleotide may have a sequence region complementary to the first oligonucleotide in the pair, as well as an arbitrary sequence nucleotide region. The first and first nucleotides are mixed under conditions such that complementary nucleic acid sequences can be bound. The first oligonucleotide can be extended in the primer extension reaction, and DNA-dependent DNA polymerase, such as reverse transcriptase, is used to synthesize the double-stranded molecules in the library, but it is not limited to this. Libraries can also be constructed using polymerase chain reactions. However, care must be taken to avoid breaking the ribose linkage under the high temperature conditions of PCR. In another way, the construction of the library can be made by chemical synthesis. As shown in Figure 1B, oligonucleotide pairs can be constructed so that the double-stranded nucleic acid molecule formed by the oligonucleotide pairs can form a hairpin loop, and the nucleotide region of any sequence can potentially interact with the cleavage site effect. In certain embodiments, the &apos; hairpin band is 6 nucleotides from the cutting position. However, it is not necessary for double-stranded nucleic acid molecules to form a hairpin loop band (see, for example, Brorraker &amp; Joyce, 1994, already 87851 -31- 200413528, which is listed as a reference to this case; see Breaker &amp; Joyce, 1995, which is listed as a full case in this case) Reference is another example of a double-stranded nucleic acid molecule including a hairpin). Without being bound by theory, Xianxin hairpins can provide advantages, so Xianxin can open up to find answers, which involves the identification of functional sites by Watson-Clike, thus popularizing the generated enzymes to different substrate sequences Can be easier. As described above, the method for identifying the catalytic DNA of the present invention usually includes administering a nicked nucleic acid that is selectively amplified by mutation at least once in a repeated selection cycle. Many methods of mutating nucleic acid molecules are known in the art. For example, mutations in nucleic acid molecules can be chemically modified to incorporate oligodeoxyribonucleic acid in any mutation, or via a polymerase incorrect grill (see Cadwell and Joyce, in PCR Methods and Applications 2: 28-33 (1992 ); Cadwell and Joyce, PCR Methods and Applications 3 (Suppl.): S136-S140 (1994); Chu? Et al.? Virology 98: 168 (1979); Shortle, et al. 5 Meth. Enzymol. 100: 457 (1983); Myers, et al. 5 Science 229: 242 (1985); Matteucci, et al.? Nucleic Acids Res. 11: 3113 (1983); Wells, et al.? Gene 34: 315 (1985); McNeil , et. al. 5 Mol. Cell. Biol. 5: 3545 (1985); Hutchison, et al. PNAS · USA 83; 710 (1986); Derbyshire, et al., Gene 46: 145 (1986); Zakour, et al., Nature 295: 708 (1982); Lehtovaara, et al ”Protein Eng. 2: 63 (1988); Leung, et al.? Technique 1: 11 (1989); Zhou, et al., Nucl. Acids Res. 19: 6052 (1991)), such as highly mutagenic PCR13. As mentioned above, the method of this aspect of the invention includes capturing one strand of a double-stranded nucleic acid molecule library. Many methods of capturing double-stranded nuclear molecules are in the art -32- 87851 200413528 The gene product can be captured or otherwise selected, such as using its ability to bind to a ligand, or to perform a chemical reaction (see, for example, Joyce Id · (1989); Robertson and Joyce, Nature 344: 467 (1990); Tuerk, et al.? Science 249: 505 (1990)) For example, a double-stranded nucleic acid molecule can include a biotin moiety so that it can be captured by a solid-phase carrier containing streptavidin. As described above, a method for identifying a catalytic DNA molecule, according to the method of the present invention, comprises expanding a nucleic acid molecule. Many methods for expanding nucleic acid molecules are known in the art. For example, nucleic acid molecules can be amplified using reciprocal primers such as polychazinase chain reaction (PCR). (See, for example, Saiki, et al., Science 230: 1350_54 (1985); Saiki, et al., Science 239: 487-491 (1988)). In addition, nucleic acid amplification can be performed by self-sustained sequence replication (3SR). (See, for example, Guatelli, et al., PNAS USA 87: 1874 (1990), whose disclosure is incorporated herein by reference). As described above, the method of identifying catalytic DNA according to the present invention generally includes growing a library of single-stranded nucleic acid molecules under conditions that allow cleavage at a cleavage site. Spring As understood by the artisan, the conditions for cutting at the cutting position depend on the specific catalytic activity required. For example, when catalytic activity refers to the ability to cleave unnaturally generated ribonucleotides, conditions may include: in three 300 microliter reaction buffer solutions (10 mM MgCh, 0.5 M NaCl, 50 mM EPPS (pH 7.5) )), At 37 ° C for 1 hour, as described in the examples. In some specific examples, as shown below, the cleavage conditions in the method may vary, so that in subsequent repetitive cleavage steps, the conditions are more efficient for nucleic acid cleavage. That is, the method of the present invention further includes: 33-87851 200413528 by changing the selection restriction in the in-tube expansion method to obtain improved catalytic properties. Therefore, as illustrated in the example, in the following cycle, such as in the 7th to 10th rounds, the reaction buffer solution can be changed to 5, ◦ call the heart like ^ mysterious sand and shorten the reaction time ^ 口 第30 minutes of the round, 5 minutes of the 8th round, 9 minutes of the 10th round. Finally, after the mutation is introduced, the reaction buffer solution used can be more urgent, such as 5mM ¥ 12'〇.15] ^ ((1 and 5〇111) \ ^? 3_7.5), and the reaction time is reduced by 11 The round of 0.5 minutes, the younger brother of 12.15 does not exceed the time required for dissolution. The present invention also proposes a set for detecting a target nucleic acid sequence, which contains a catalytic DNA molecule with a non-naturally occurring ribonucleotide and / or an acceptor set, which may include: a primer, a polymerase, and Other reagents useful in the methods of the invention. In some specific examples, the set of nucleic acid sequences to be detected by the present invention contains: a single-stranded nucleic acid sequence that is not naturally generated as described above, a forward primer and a reverse primer, which include the aforementioned catalytic A single-stranded DNA molecule complement, in which a catalytic single-stranded DNA molecule can cleave a acceptor nucleic acid sequence at a cleavage site. The kit can include duplexes, where the non-naturally occurring ribonucleoside cleavage site includes 2 ', 5' contiguous residues. In other specific examples, the kit may include a literary substance, wherein the non-naturally occurring ribonucleoside cleavage position includes an L-enantiomeric residue. The following examples are used to illustrate but not limit the invention. Example 1

87851 -34- 200413528 The following examples illustrate the use of direct expansion to produce and isolate DNA enzymes, which can cleave non-naturally occurring ribonucleotides and the identification of isolated DNA enzymes. The enzymes illustrated in this example can cleave the acceptor nucleic acid sequence at 2,5'-acid diester after D-ribonucleotide, or 3 ', 5f- after cleaving L-ribonucleotide. Phosphodiester. * Experimental paragraph Chemical synthesis of oligonuclear acid. Tertiary-butyl-dimethyl sulfanyl- and (3-L-2'-tertiary-butyl-dimethylsilyl-ribonucleoside aminophosphite) from ChemGenes (Ashland, MA ), And all other nuclear amino amino linoleates were obtained from Glen Research (Sterling, VA). All oligonucleotide acids were prepared by automated synthesis using an Applied Biosystems Expedite Nucleic Acid Synthesizer. Butyl-dimethylsilyl and L-ribose riboamidophosphite are coupled in a 15-minute coupling step. The resulting oligonucleotides are incubated in anhydrous saturated NH3: ethanol at 37 ° C for 36 hours Deprotection was followed by incubation overnight in a 1 M solution of tetrabutyl neodymium fluoride in THF at room temperature. All other oligonucleotides were synthesized and deprotected using standard procedures. All purification of oligonucleotides All were subjected to denaturing polyacrylamide gel electrophoresis (PAGE) and desalted on NAP-25 column (Pharmacia Biotech, Piscatway, NJ). 4 Nanomolar 5'_Biotin- d (TTTTAGAGACGATGACG ATGCAXTCGGACAGTCGCGAGACTGV3, (SEQ ID NO: 23)

(Primer 1; X = 2 ', 5'-rG or Li: A) at 6 nanomolar 5'-d (GTGCCAAGC TTACCG-Nn-CAGTCTCGCGACT GTCCGAV3, (SEQ ID 87851 -35- 200413528 No.:24) (N = A, C, G, or T; underlined complementary sequences) The upward extension reaction can construct a starting pool of ~ 1015 DNA molecules. 1 ml of the reaction mixture contains 5 units / microliter of Superscript II reverse transcriptase ( Life Technologies, Gaithersburg, MD), 3 mM MgCl, 75 mM KC1 '50 mM (via methyl) -aminomethane (Tris5 pH 8.3), and 0.25 mM dATP, dGTP, dCTP, and TTP extension reactions The two oligonucleotides were cooled back at 85 ° C for 4 minutes, cooled to room temperature, added MgCl2 and reverse transcriptase, and incubated at 37 ° C for 1 hour. The extension product was purified by undenatured page Dissolve from the gel, immerse in ethanol, and re-dissolve in 1 ml containing 1-2 μM extension product, 0.5 M NaCL ·, 0.2 mM Na2EDTA, and 50 mM Ν- (2-hydroxyethyl)- Piperazine-N, 3-propanedioic acid solution (EPPS, pH 7.0). This substance was applied to an affinity column containing 300 microliters of UltraLink Immobilized Streptav idin PLUS gel (Pierce, Rockford, IL) and pre-equilibrated with four 400 μl wash buffer solutions (0.5 MNaC, 0.1 mM Na2EDTA, 50 mM EPPS (pH 7.0)). The column was divided into five 400 microliters of wash buffer solution, five 400 microliters of ice-cold 0.1 N NaOH / 150 mM NaCl and five 400 microliters of wash buffer solution, rinse for 1 hour at 37 ° C, and then at 37 ° C Dissolve in three, three hundred microliters of reaction buffer solution (10 mM MgCl2, 0.5 M NaCl, 50 mM EPPS (pH 7.5)). The molecules leached from the column are ethanol, and the primers are 150 μM 5 ' -d (TCGGACAGT CGCGAGACTG) -3 '(SEQ ID NO: 25) (primer 2) and 250 picomolar primer 5, -d (AACA ACAACYYYGTGCCAAGCTTACCG) -3, (SEQ ID NO: 26) (primer 3; none Basic nucleic acid analogs) were precipitated and dissolved in the presence of 500 μl of PCR amplification reaction. Three non-basic analogs can generate a stop position for the Taq polymer 87851 -36- 200413528 enzyme, which causes one of the PCR product strands to be 12 nucleotides shorter than the other. The enlarged product was precipitated with ethanol, the longer of the two strands was separated by denaturing PAGE, dissolved from the gel, and precipitated again with ethanol. Take half of the dissociated DNA (~ 80 picomoles) and apply it to the template-command extension reaction, where 200 picomoles primer 1 is applied under the same conditions as above. In this and all subsequent selective expansion cycles, the extension product is solidified on 50 microliters of Streptavidin Plus gel, five 200 microliters of wash buffer solution, and five microliters of 200 microliters of ice-cold 0.1 N NaOH / 150 mM NaCl and five 200 microliter wash buffer solutions were rinsed at 37 ° C, and then dissolved in three 40 microliter reaction buffer solutions for 1 hour. In the second round, the molecules to be reacted can be additionally selected based on the electrophoretic moving force on the denatured polyacrylamide gel. In the 7-10 round, the reaction buffer solution was changed to 5 mMMgCl2, 0.2M NaCl and 50 mM EPPS (pH 7.5). The reaction time can be reduced to 30 minutes in the seventh round, and 5 minutes in the eighth round. The ninth and tenth rounds are minutes. After the tenth round, arbitrary mutations can be introduced by PCR13. Perform another five rounds of selective expansion response, in which the reaction buffer solution was changed to 5 mMMgCl2, 0.15 MNaCl and 50: mM EPPS (pH 7.5), and the reaction time was reduced to 0.05 minutes in the u-th round. , And in the 12-15 round is not more than the time required for dissolution. Analysis of individual pure lines

After the 10th and 15th rounds, the DNa molecule was enlarged with pCR, and primers 2 and 3 were used. The truncated version has the sequence 5, _d (GTGCCAA GCTTACCG) -3 '(SEQ ID K0: 27). PCR products were cloned using the TA colony kit and INVaF 'competent cells (Invitrogen, Carlsbad, CA). Individual pure 87851 • 37-200413528 were isolated on agar plates and expanded by colony PCR or 2 ml culture inoculation. DNA was isolated and sequenced by dideoxy chain termination method 14. The cut analysis was performed under conditions similar to those applied in the test tube selection. The reaction was stopped by adding an equal volume of a mixture containing 10 M urea and 50 mM Na2EDTA, and the reaction products were separated by denaturing PAGE and analyzed by Molecular Dynamics Phosphorimager. Kinetic analysis In the presence of 25 mM MgCl2, 150 mM NaCl and 50 mM EPPS (pH 7.5), an intermolecular cleavage reaction was performed at 37 ° C. The start of the reaction is the addition of substrates to enzymes, each corresponding to the final reaction buffer solution in the mixture. The reaction products were separated by denaturing PAGE and analyzed by Molecular Dynamics Phosphorimager. The Us value can be obtained under a single conversion (enzyme excess) condition, in which various concentrations of enzymes and trace amounts of [5'-32P] are labeled substrates. The experimental data of L-ribonucleic acid-cleaving DNA enzymes are combined into the single-finger equation: F ^ Fyi-e — ^ + Fo, where A is the part that cuts at time t, and it cuts to the maximum extent Part, and R is the part cut at zero time. The experimental data of 2 ′, 5’-ribonucleic acid cleavable DNA enzymes are combined into the double exponential equation:

Ft = F1 (l ^ ekobsht) + F2 (l ^ ekobs2 ^) + F〇 ^ where Fi is the part cut at time t, A and A are the amplitudes of the two phases of the reaction, and ^^ 2 is the equivalent rate of each phase , And A is the part cut at zero time. These variables are estimated using 1 ^ ¥ 61 ^ 6 [8-] \ ^ 1 ^ 1 ^ 1 (11: algorithm (〇61138 to 114.5, SPSS Science) with non-linear regression. 87851 -38- 200413528 The value of ^^ can also be obtained under the condition of excess mass, where the concentration range of the mass spans. Before the reaction, more than 15% of the data can be obtained, and the data are combined on a straight line. It is usually based on six data points. In the standard Michaelis_Menten saturation plot consisting of u-21 data points, the variables [M and 4] can be obtained, and [S] is always at least 10 times greater than [E], and [E] is at least 5 times lower than the threshold. Adjustment The data should take into account the maximum reaction level for L-ribose ribonate-cleaving enzymes, and the amplitude of the first phase of the reaction for cleavage of DNA enzymes with 2,5, -ribonucleotides. Using SigmaPlot (SPSS Science) to calculate the standard error value. Incubate 1 nM [5, -32P] -labeled substrate under standard reaction conditions to determine the rate of uncatalyzed cutting. Take out each portion during 5 days to denaturate 1> 80. £ analysis. From the slope of the most suitable straight line plot of the cut part versus time, eight kuncat can be obtained. Metal, temperature and pH Dependence. All values were obtained under single conversion conditions, where 90 × M enzyme and 1 nM [5, _32P] -labeled substrate were used, which were cultured under standard reaction conditions as described above. MgCL dependency assessment, Uoo concentration range is used for 2 ', 5'-ribonucleic acid-cleaving DNA enzymes, and o.uomM is used for L-ribonucleotides and cleavage DNA enzymes. 10 mM M2 + is necessary for testing metal ions, but The pb2 + exception is tested at a concentration of 1 mM. The temperature dependence is determined in the range of 10-651, using a temperature plate and a heating cover to control evaporation at high temperatures. The pH dependence is evaluated in the range of 6.0-9.5, using three Different buffering substances; 2_ [N-morpholinyl] -Ethyl carbamic acid (MES), at pH 6.0-7 · 0, EPPS at pH 7 0-8 5, and cyclohexylamino] -ethanesulfonate Acid (CHES) at pH 8.5-9.5. Identification of cleavage products 87851 -39- 200413528 For large-scale reactions, using 1 nanomolar substrate containing 2 ', 5'- or L-ribonucleotides, Add 1 nanomolar equivalent DNA enzyme under the above standard reaction conditions. After 24 hours, the reaction is stopped and the product is cleaved to Purification by denaturing PAGE. Prior to the reaction, the receptor containing L · -ribonucleic acid is T4 polynucleotide kinase and ATP 5'-phosphorylated. In this way, two 9 members can be separated by their different electrophoretic mobility. The product was cut. The gel-purified product was desalted on a Nensorb-20 column (NEN Life sciences) and analyzed by MALDI-TOF mass spectrometry, where a PerSeptive Biosystems Voyager-STR mass spectrometer was used. Result-In-tube selection. Construct two separate libraries of ~ 1015dna molecules, one containing a single 2 ', 5f-linked guanine nucleotide ribonucleotide, and the other a single L-adenosine Acid ribonucleoside, embedded in the whole DNA sequence of other aspects. The library is constructed by a primer extension reaction, using 5'-biotinylated primers containing non-natural ribonucleotide linkages. The DNA template to which the primer hybridizes contains 50 deoxynucleotides of any sequence, flanked by residues of a defined sequence, which can be used as a primer binding site. Similar to the previously applied strategy, 15 DNA hairpins are genetically manipulated into molecular pools to facilitate base-pair interactions around the target ribonucleoside analogs (Figure 1B). A primer extension reaction is performed using a reverse transcriptase, which acts as a DNA-dependent DNA polymerase to produce a double-stranded product. In the primer extension reaction, since the two strands maintain a duplex, no DNA-catalyzed cleavage reaction occurs. The full-length double-stranded product was purified by non-denaturing PAGE and quantified based on its UV absorbance. The purified material is solidified on a solid 87851 -40-200413528 phase carrier containing streptavidin, and the non-biotinylated strand is quickly washed and removed with an ice-cold solution of 0.1 N NaOH. Biotinylated single-stranded molecules still remain bound to the carrier, and then challenged to cleave the embedded ribonucleotide linkage, which can be released from the carrier. Initially, the reaction conditions chosen were favorable for the formation of double strands, with high salt concentrations of 10 mM MgCl2 and 500 mM NaCl, at pH 7.5 and 37 ψ ° C. The released molecule collection was amplified by PCR, thus enriching the population of reactive molecules. A total of 15 rounds of selective expansion reactions were carried out to obtain the most active catalyst. In the first six rounds, the reaction conditions were as described above, and the reaction time was 1 hour. In 7-10 rounds, the reaction conditions were changed to 5 mM MgCl2 and 200 mM NaC at pH 7.5 and 37 ° C. In rounds 7 and 8, the reaction time can be reduced to 5 minutes to increase the urgency of choice; in rounds 9 and 10, the time is further reduced to 1 minute. After the 10th round, individual molecules were selected from the population, then sequenced and tested for catalytic activity. The population was mutated arbitrarily, with a frequency of ~ 10% per nucleotide position, and another 5 cycles of selective expansion were performed, using 5 mM MgCl2 and 150 mM NaCl, pH 7.5 and 37 ° C reaction conditions. The reaction time is 0.5 minutes for the 11th round, and the time required for dissolution is not more than 12-15 rounds. Individuals were re-selected from the population and sequenced, showing a high degree of similarity of sequences within any previously sequenced region (see supplementary material). Identification of catalytic motifs. One of the selected individuals isolated after selection of the 2 ', 5'-field acid diester-cleaving activity in the 10th round had a particularly high activity, and was selected for further research. Its fate is 'f2f: 10-16', because it is the 16th pure line of facts separated after the 10th round. The sequence analysis can infer that there is a reasonable secondary structure, as shown in Figure 2A The enzyme can be prepared separately and affected by the 87851 -41 200413528 mass strand 'system to extend the reaction to the putative base pair region around the cleavage position, and repair any base mismatches. For 2,5, -phosphodiester_ Cleavage activity. The selected individuals isolated after the 15th round of selection have approximately the same level of activity as the 2 :: 10-16 pure line. In the pure lines isolated after the 15th round, it can be noted that the sequence similarity is extremely high. (See Figure 9A). A representative pure line with a life of, 2 \ · 15-2, was selected for further analysis. It was prepared through chemical synthesis, and the enzyme and receptor stock were separated in the intermolecular reaction pattern. A cleavage phenomenon can occur (Figure 2b). As will be described in detail below, 2, ·· 1 (M6 and 2: 15_2 DNA enzymes can cut as much as 1 ~ 1 minute 1 and separate the substrates. In efforts When confirming the 2 ', 5, -phosphodiester_ cleavage of the inferred secondary structure of DNA enzymes, Inside the unpaired region, also referred to herein as internally bulging loops (see Figure 15), various nucleotide substitutions and deletions are made, most of which can result in a total loss of catalytic activity. Two internal bulges are shown The inferred flanking region on either side of the loop belt can be interchanged with any or base pair &lt; nucleotides that have little or no effect on the catalytic rate. When the inner loop belt is furthest from the cutting position Also referred to as the upstream loop zone here, it can be replaced by a single τ residue, forming a contiguous segment downstream of the cleavage site, and the catalytic activity is eliminated. However, this inner swelling loop zone may be somewhat Alterations using 3, -AGGGATTTG-5, (Figure 2A) '3-AGGG-5' (Figure 2B), 3, -AGGGATTCG-5, and 'can all result in complete catalytic activity. Finally, on the receptor molecule The unpaired G residues immediately upstream of the cleavage position can be changed to live ... / and reduced, but when changed to .or u, it results in a complete loss of activity. For the cleavage activity of ribonucleic acid, 878 87851 -42- 200413528 separated after the 15th round of selection After selecting the individual, and after the 10th round of selection, The isolates are more active. The pure lines that were separated after the last round also have a high degree of sequence similarity. (See Figure 9b). A representative pure line, named "L: 5_3〇", was selected for further analysis. It is prepared by chemical synthesis, separating enzymes and substrates, extending the base pairing region inferred around the cleavage position, and repairing any base mismatch (Figure 2C). At 2 ', 5'-phosphodiester -In terms of cleaved DNA enzymes, the base paired nucleotides around the cleaved position can be replaced by any little or no effect on the catalytic rate &lt; any paired nucleotides. Mutation analysis is no longer performed on the ribonucleotide cleavage motif. Biochemical properties of DNA enzymes. The catalytic properties of the above three DNA enzymes were all studied in the intermolecular reaction pattern (Figure 2A_C). Time_flow experiments show that 2 ′: 10-16 and 2: 15-2 DNA enzymes exhibit two-phase kinetics, starting with a fast onset rate and followed by a slower reaction in the second phase (Figure 3). Therefore, the data is delicious. I double the number equation and determine the catalytic rate constant for each phase of the reaction (see the experimental section). In contrast, the L: 15_30 DNA enzyme exhibits a single-phase kinetics, which is very close to the single exponential equation (Figure 3). Under multiple conversion conditions, the product is released as rate-limiting all 2,10-6 and 2 ': 15-2 DNA enzymes. This can be confirmed by comparison of multiple conversion reactions and single conversion reactions under conditions of excess enzymes, the latter being about 10 times faster. On either or both sides of the cleavage position, the release of beneficial products from the base-pairing region between the enzyme and the substrate is shortened. However, the release of the product is still rate limiting until the paired regions are shortened to impaired catalytic activity (data not shown). The highest value is obtained when the 2 :: 10-16 enzyme is shortened by one base pair at each end of the enzyme-receptor complex. Under multiple conversion conditions, this construct exhibited a value of 〇0036 ± 〇〇〇〇 87851 -43-200413528 minutes, and 021 ± 0 03 [the value (Figure 4B). By comparison, the full-length construct exhibits a [value-added value of 0.0002 ± 0.0001 minutes-1, and a value of 0.0042 ± 0.008 riM (Figure 4A). Under the condition of excessive enzymes, the full-length cutting rate is 2 ′: 10.16 enzymes are 0.001 11 ± 0.0004 minutes (Figure 5A), which is 5 times higher than that obtained under multiple conversion conditions. The 2 :: 15_2 enzyme was slightly faster than the 2 ′: 1〇-16 enzyme under both single and multiple conversion conditions. 2 :: 15-2 [cai value: 0.0012 min 0.0004 min-1 and 0 064 ± 〇09 hit l value (Figure 4C). This corresponds to a catalytic efficiency of ~ 108 NT1 min · 1, ‘, / 1. Under the conditions of excess enzyme, the rate of 2: 15-2 enzyme is 〇034 ± 〇0011 minute-ι (Figure π), which is about 3 times higher than that obtained under Douer conversion conditions. The base-pairing region between L: 15-30 DNA enzymes and their substrates can be shortened, so the release of this product is no longer rate-limiting and does not cause a reduction in catalytic rate. Under multiple conversion conditions, the shortened enzyme exhibited Michaeiis-Menten saturation kinetics, U = 0.0012 ± 0.0001 min-1, and the 'value was 2.9 ± 0.8 11] \ 1 (Figure 4). In the case of enzyme excess, the catalytic rate is 0.0001 ± 1.001 min-1 (Fig. 5C), which is very similar to the special cai value obtained under multiple conversion conditions. For 2 ': 1 (M6 and L: 15-30 DNA enzymes, explore the effects of temperature, pH, and Mg2 + concentration on DNA-catalyzed reactions under single conversion conditions. The optimal temperature for the two enzymes is ~ 42 ° C, slightly higher than the temperature used in the selection process in the test tube (see supplementary materials). 2,: 1 (M6 DNA enzyme is most suitable for pH of about 7.5, below pH 6 · 5 and pH 8 · 5 The above decreases the activity (Figure 6A). L: 1 5-3 0 The catalytic rate of DNA enzymes is pH-independent in the range of ρΗ 6.0-9.0 (Figure 6B). It is inferred that the reaction rate determines the step (not a chemical step). 87851 -44- 200413528 2 ': 10-16 and L: 15-3 0 The catalytic rates of DNA enzymes are related to the concentration of Mg2 +, and both show saturation behavior in two cases. 2 :: Appearance of 10-16 enzymes

A ^ (Mg2 +) IKNF ~ 4 mM, MBLL L.15-30 DNA enzyme is ~ 0.6 mM (see Figure 10). Test the ability of many different divalent metal cations to support 2, 10-16 and L: 15-30 DNA enzymes to catalyze the reaction. 2: 1 (M6 dnA enzyme in

In the presence of Mg, the activity level of the south is shown, and the activity gradually decreases in the presence of ca2 +, ^ 2 +, or Ba2 +, and there is little or no activity in the presence of Mn2 +, Pb2 +, cd2 +, and Co2 +. L · · 15-30 DNA enzyme is most active in the presence of Mn2 +, and its activity gradually decreases in the presence of Mgu, Ca2 + or Pb2 +, and there is little or no activity in the presence of Ba2 +, Sr2 +, Cd2 +, Co2 + or Zn2 + (see Figure 丨 2) . None of the enzymes showed activity in the presence of co (NH3) 6 (data not shown). The high-resolution PAGE and MALDI mass spectrometry were used to analyze the cleavage products obtained from the reaction under the enzymes 2: 10-16 and 15-30. In two cases, the 5, _ cleavage product is an oligonucleotide of the expected length, which terminates at 2,3, _cyclic phosphoric acid or 2, _ or 3, _monoscale bismuth (see Figures 7 and 13) . The 3'-cleaved product also has the expected length and terminates in the free 5'-hydroxyl group, which can be confirmed by MALDI mass spectrometry (see Figure 13). Determine the 2,5, _phosphodiester cleavability and 1_ribonucleic acid-the region of cleavable DNA enzymes-and the enantiomerism specificity under single conversion conditions, and compare the non-natural or natural Substances of ribonucleic acid (compare Figures 3 and 8). Various substrates can also be used to detect uncatalyzed cutting rates (see Figure 14). For 2,5, -linked ribonucleotides, 2,: 15-2 DNA enzymes present ~ 20,000 ft / U straight, while 3,5, _- linked ribonucleosides are quite substrate There is 3.3 especially, reflecting that the regional selection of 87,851-45-200413528 is about 6,000 times more selective than non-natural receptors. 2f: 10-16 The area of DNA enzymes is approximately 2,000 times more selective. L: 15-30 DNA enzymes are not as selective as 2 ′, 5 ^ cleavable DNA enzymes. For substrates containing L-ribonucleic acid, such as about 500, and for substrates containing D-ribonucleotides, increase by about 13. This is equivalent to about 40 times the enantioselectivity of non-natural substrates. In the autoradiogram of Fig. 7, the 2 ', 5'-phosphate- or L-ribonucleic acid-region of cleavable DNA enzyme-or enantioselectivity is shown clearly. Discussion Enzyme specificity of an enzyme is determined by its ability to discriminate between substrate-binding steps and chemical reactions. For small molecule substrates, it is more difficult to achieve a high degree of differentiation compared to large molecule substrates, because the number of possible interactions between enzymes and small molecules is less. The interaction between the two nucleic acid molecules can be highly specific, subject to sequence confirmation involving Watson-Crick base pairing and non-standard pairing interactions. Nucleic acid molecules have also been explored for their ability to distinguish one another based on their region- or enantiomeric composition. For example, 2 ', 5'-linked RNA can form a stable double strand with 2', 5'- or 3 ', 5'-linked RNA, but 3', 5'-linked DNA is not 16 . Similarly, 2 ', 5'-linked DNA can form a stable double strand with 3f, 5f-linked RNA, but 3', 5'-linked DNA is not17. All-L-oligodeoxynucleotide consisting of six guanine nucleotide residues has been shown to be paired with complementary all-D-RNA strands, but not comparable to all-D-DNA18. However, another study reported that neither all-L-DNA nor all-D-RNA or all-D-DNA could form double strands19. In this study, the substrate contains a single unnatural ribonucleotide and is embedded in another all-unnatural DNA molecule. It presents a more difficult challenge for the specific confirmation of the region- or enantiomer 87851 -46- 200413528. . The DNA enzyme disclosed in this example is highly specific for receptors containing a single non-natural ribose ribonucleic acid. An enzyme distinguishes 2'55, _ and 3,, 5, -linked residues with a region specificity of 6,000 times. The other selectively distinguishes L- and D-residues by 40-fold enantiomers. The catalytic rate of the phosphodiester-cleavable DNA enzyme is ~ 0.01 min-1. The L-ribonucleic acid-cleaving DNA enzyme is about 10 times slower. These rates are significantly slower than other reported RNA-cleaving DNA enzymes that cleave natural ribonucleic acid23. For example, 10-23 '' DNA enzymes can reach catalytic rates of up to min-1 under the most suitable reaction conditions24. It may be possible to cleave non-natural ribonucleic acid DNA enzymes, which is difficult to fold into an active configuration, or put a divalent metal ionization to help the cutting of the target phosphate diester. Similar to known ribonucleases, the cleavage mechanism appears to involve the deprotonation of the free state 2, _ or 3, _ hydroxyl groups, and the oxygen anions generated after the protonation reattach to the adjacent phosphate. For this adhesion, there needs to be a line of orientation, which can be achieved by forcing the nucleotides out of the helix before the cleavage position25. A single unpaired purine nucleotide in another fully double-stranded structure is particularly easy to achieve with this orientation via a local configuration change, which mainly involves the% of torsion angle of the ε &amp; ζ skeleton. In order to achieve the desired orientation within the Watson-Crick double strand, it may be difficult to bind the linked D-ribose riboic acid, and especially the 3,5, _ linked ribonucleoside bismuth. Therefore, in order to complete the cleavage of these non-natural ribonucleosides, additional catalytic assistance is required. The uncatalyzed cleavage rate of 3 ', 5'-phosphodiester in RNA is for single ribonucleotide 27 embedded in another all-DNA molecule, and for all-RNA oligos μ 87851 -47- 200413528 both Take measurements. The hydrolysis rate of phosphoric diesters in RNA without catalysis is similar to 3'5'-phosphodiesters 12′29_32 in the presence or absence of divalent metal cations, except when the RNA is bound to complementary strands. In this example, the 2 ', 5'-linkage is about 7 times more unstable, and the 3', 5'-linkage is about 5 times less stable33. In order to determine whether this "double-stranded effect" should respond to the noble task of enhancing the negative part of the catalytic rate observed in 2f, 5'-cleaving DNA enzymes, it will contain 2 ', 5'-linked ribonucleosides The substrate of the acid hybridizes with the complementary DNA strand, and the hydrolysis rate is compared with the hydrolysis of the substrate alone. Under these two conditions, there is no difference in the uncatalyzed cleavage rate. (Data not shown). For single embedding The uncatalyzed cleavage rate measured by ribonucleic acid is similar to the previously reported 14'28. The DNA enzyme developed in this study does not essentially have any enantiomeric moiety and is known by any known None of the biological receptors can function. However, it can be used as a useful biochemical tool in cutting the informer molecule containing non-natural ribonucleotide and acid. This informer cannot be cut by biological nucleases. This activity has a The potential application, as detailed above, is a method for quantitative PCR, called MDzyNA-PCR''34. In this method, RNA-cleaving DNA enzymes are used to cleave-informer oligonucleotides, and their Tie on either side of the cutting position There is a fluorescent marker and a cooling agent. The sequence of the DNA enzyme is encoded by a complementary sequence that is adhered to the 51 end of one of the two PCR primers. When PCR amplification is performed, it can generate a set of functional DNA enzymes. It can cut The informer molecule separates the fluorescent marker and the quencher to generate a fluorescent signal. In the existing method, because the informant contains natural ribonucleic acid, it is susceptible to cleavage by biological ribonuclease. However, if This is not the case when we use DNA enzymes that can cleave informants that have non-natural ribonucleotides in the molecule. 87851 -48- ^ 528 There are naturally occurring ribonucleases that cleave 2, 5 »_link The production of these molecules is part of the interferon response pathway. However, 'for 2', 5, the cleavage of linked guanine ribonucleic acid or ^ ribonucleic acid 'has no known organisms. In addition to its potential application as a biochemical tool, the DNA enzymes described here indicate that the acceptor region_ and enantioselectivity of nucleic acid enzymes are better than their natural protein counterparts. Servings Snake venom structure acid diesterase I can cleave D- or L-ribonucleic acid, but it is 1,800 times more active in cleaving natural d-RNA substrates. 36 It is then expanded in further test tubes In experiments, especially those with enhanced potency nucleic acid analogs 37,38, novel catalysts with even larger regions or enantioselectivity will be developed. 87851 49- 200413528 References (1) Joyce , GF; Visser, GM; van Boeekel, CAA; van Boom, JH; Orgel, LE; van Westrenen, J. Nature 1984, 310, 602-604. (2) Kozlov, IA; Pitsch, S .; Orgel, LE Proc. Natl Acad, Sci, USA 1998, 95, 13448-13452. (3) Saghatelian, A .; Yokobayashi, Y .; Soltani, K .; Ghadiri, MR Nature 2001, 409, 797-801. (4) Breaker , RR Chem. Rev. 1997, 97, 371-390. (5) Jenne, A .; Famulok, M. Topics Curr. Chem. 1999, 202, 101-131. (6) Petrounia, IP; Arnold, FH Curr Opin. Biotechnol 2000, 77, 325-330. (7) Kolkman, JA; Stemmer, WPC Nature Biotechnol 2001, 19, 423- 428.-(8) Reetz, Μ. T. Angew. Chem. Int. Ed. Engl 2001, 40, 284 -310. (9) Seelig, B .; Keiper, S .; Stuhlmann, F .; Jaschke, A. Angew, Chem, Int Ed, Engl 2000, 39, 4576-4579. (10) Bartel, DP; Szostak, JW Science 1993, 26191411-1417. (11) Ekland, EH; Szostak, JW; Bartel, DP Science 1995, 269, 364-370. (12) Shih, L-Η .; Been, MD RNA 1999, 5, 1140 -1148. (13) Vartanian, J.-P .; Henry, M .; Wain-Hobson, S. Nucleic Acids Res. 1996, 24, 2627-2631. (14) Sanger, F .; Nickelson, S .; Coulson, AR Proc. Natl Acad. Sci. USA 1977, 74, 5463-5467. (15) Breaker, RR; Joyce, GF Chem. Biol. 1995, 2, 655-660. (16) Wasner, M .; Arion , D .; Borkow, G .; Noronha, A ·; Uddin, A. H ·; Pamiak, M · A ·; Damha, M · J. 1998, 37, 7478-7486 · (17) Alul, R. ; Hoke, GD Antisense Res. Dev. 1995, 5, 3-11. (18) Fujimori, S .; Shudo, KJ Am. Chem. Soc. 1990, 1129 7436-7438. (19) Garbesi, A ·; Capobianco , M. L .; Colonna, F. P .; Tondelli, L .; Arcamone, F .; Manzini, G .; Hilbers, C. * W .; Aden, J. Μ. E .; Blommers, MJJ Nucleic Acids Res. 1993, 27, 415 9-4165. -50- 87851 200413528 (20) 4266. Santoro, S .; Joyce, GF Proc. Natl. Acad. Sci. USA 1997, 94, 4262- (21) 282. Sugimoto, N .; Okumoto, Y . Nucleic Acids Symp. Series 1999, 42, 281- (22) 481-488. Li, J .; Zheng, W .; Kwon, AH; Lu, Y. Nucleic Acids Res. 2000, 28, (23) Feldman, AR; Sen, DJ Mol. Biol. 2001, 313y 283-294. (24) Santoro, S .; Joyce, GF Biochemistry 1998, 37, 13330-13342. (25) Husken, D .; Goodall, G .; Blommers , MJJ; Jahnke, W .; Hall, J .; Haner, R .; Moser, Η. E. Biochemistry 1996, 55, 16591-16600. (26) Tereshko, V .; Wallace, ST; Usman, N .; Wincott, FE; Egli, M. RNA 2001, 7, 405-420. (27) Jenkins, LA; Autry, M. E .; Bashkin, JKJ Am. Chem. Soc. 1996, 118, 6822-6825. (28 ) Li, Y .; Breaker, RRJ Am, Chem. Soc. 1999, 121, 5364-5372. (29) Oivanen, M .; Kuusela, S .; Lonnberg, H. Chem. Rev. 1998, 98, 961- 990. (30) Oivanen, M .; Schnell, R .; Pfleiderer, W .; Lonnberg, HJ Org. Chem. 1991, 56, 3623-3628. (31) Jarvinen, P .; Oivanen, M .; Lonnberg, H J. Org. Chem. 1991, 56, 5396-5401. (32) (33) 1149-1153. Anslyn, E .; Breslow, R. J, Am. Chem. Soc. 1989, 111, 4473-4482. Usher, DA; McHale, AH Proc. Natl Acad. Sci. USA 1976, 73, (34) Todd, AV; Fuery, CJ; Impey, HL; Applegate, TL; Haughton, MA Clin. Chem. 2000, 46, 625 -630. (35) Nilsen, TW; Wood, DL; Baglioni, C. 1 Biol Chem. 1982, 257, 1602-1605. (36) 1484. Moyroud, E .; Biala, E .; Strazewski, P. Tetrahedron 2000, 56, 1475- (37) Santoro, S .; Joyce, GF; Sakthivel, K .; Gramatikova, S .; Barbas, CFJ Am. Chem. Soc. 2000, 122, 2433-2439. (38) Perrin, DM; Garestier, T .; Helene, C. J, Am. Chem. Soc. 2001, 123, 1556-1563. -51- 87851 200413528 Although the present invention has been described with reference to the above examples, it should be understood that modifications and variations are covered It is within the spirit and scope of the present invention. Therefore, the present invention is limited only by the following patent claims. [Brief Description of the Drawings] Figures 1A and 1B show compounds used to develop DNA enzymes, which can cut non-natural ribonucleic acid analogs. Fig. 1A shows the chemical structure of 2,5, · -linked bird's throat nucleic acid (left), and 3,5, _- linked p-L_adenine nucleotide (right). Figure 1B diagram Structure of a DNA molecule starting library for obtaining a desired DNA enzyme. Each molecule contains a 5, terminal biotin (closed B), and at the target cleavage position (χ) is 2, 5, 5, linked D_ ribonucleotide or 3 ', 5'-linked L- Ribonucleic acid, downstream of the cleavage site, is a fixed hair band region (shown as a sequence) (SEq ID n0: 31) and 50 arbitrary sequences of deoxyribonucleic acid (N50). Figures 2A-2C show 2 ': 1 (M6 (SEQ ID NOS: 32,9) (Figure 2A), 2 :: 15-2 (SEQ ID NOS: 32,10) (Figure 2B), and L: 15-30 (SEQ ID NOS: 33, 18) (Figure 2C) The inferred secondary structure of the catalytic DNA molecule, each showing its binding to the substrate in an intermolecular reaction pattern. The bold letters 〇 or 入 show 2 respectively. , 5, _ linked β-D-uridine nucleotide or 3,5, _ linked L_adenine nucleotide. The arrow shows the cutting position. Figure 3 -16 (filled circles), 2: 15-2 (filled squares), and the time course of the cleavage reaction catalyzed by L · 15-30 DNA enzymes (filled triangles) are measured under single conversion conditions. Illustration Shows the details of j, 000 minutes before the reaction, showing the two-phase nature of two 2 ', 5'_phosphodiester-cleaving enzymes. Data can be combined into single or double exponential equations (see experimental section of the example) Parts). The reaction was 87851 -52- 200413528 under the conditions of 25 mM MgCl2, 150 mM NaCl, pH 7.5 and 37 ° C. Figures 4A-4D show the catalytic activity of an ONA enzyme that can cleave non-natural ribonucleotide analogs, Under multiple conversion conditions. (Figure 4A) 2: 1 (M6 DNA enzyme has a full-length stem area around the cutting position; (Figure 4B) 2: 10-16 DNA enzymes each have a base pair short in the stem area; (Figure 4C) 2 ': i5 -2 DNA enzyme '(Figure 4D) L: 15-30 DNA enzyme. The data are combined in a curve based on the Michaelis-Menten equation: v = u substrate] [substrate]). Reaction conditions: 25 111] ^] \ ^ 0: 12, 15〇111] \ 4 cents? 117.5 and 37. (:. Figures 5A-C show that the catalytic activity of the DNase enzyme that can cleave non-natural ribonucleic acid analogs is β 'under single conversion conditions. Determination. It is determined under various enzyme concentrations, and the data are combined with the following equations: dagger U enzyme] / (A + [enzyme]), where at a saturated concentration, and A is the apparent cleavage constant of the enzyme receptor complex. (Figure 5A) 2 ': 10-16 DNA enzymes, such as moxibustion of 〇11 soil 0.0004 minutes 1' A is on ± 0 · 01 nM; (Figure 5B) 2 ': 15-2 DNA enzymes' free addition is 0.034 ± 0.001 min-1, and the heart is 0.12 ± 0.011; (Fig. 5 (3) L: 15-30 DNA enzyme, m1616 ± 0.001 min- 1 and the heart is 3 2 soil 0.5 nM. Reaction conditions: 25 mM MgCl2, 150 mM NaCl, pH 7.5 and 37 ° C. Figures 6A-6B show the pH dependence of DNA-catalyzed reactions. (Figure 6A) 2f: 10-16 DNA enzyme; (Figure 6B) L: 15_30 DNA enzyme. The buffer solution is MES (circle), EPPS (square) or CHES (triangular). Fig. 7 shows an autoradiogram, which is a cleavage reaction catalyzed by L: 15-30 DNA enzyme or 2: 10-16 DNA enzyme, each of which is substrated by its equivalent unnatural ribonucleotide, or where Natural ribonucleotides were replaced by the standard ribonucleotides 87851 -53-200413528. Reaction conditions: 25 mMMgCl2, 150 mM Naa, and 37 C, and incubated for 6 hours in the presence or absence of DNA enzyme (+). The non-natural ribonucleotide substrate also undergoes alkaline hydrolysis of plutonium, which was cultivated in the presence of 0.1 N NaOH for 6 hours at 37 ° C. Fig. 8 shows the kinetics of cleavage catalyzed by plasmid DNA, which contains a natural ribonucleic acid in place of unnatural ribonucleic acid. The reaction was performed in the presence of a saturated concentration of DNA enzyme, in which 2 :: 10-16 DNA (circle), 2f: 15-2 DNA enzyme (square) or 1: 15_30 DNA enzyme (triangle) were used. The catalytic rate is obtained from the most straight line plot of the data as a function of time. Reaction conditions: 25 mM MgCl2, 150 mM NaQ, pH 7.5 and 37 ° C. 9A-9B show the sequence of individual pure variable regions (SEQ ID NOS 1) isolated for 2, [[phosphodiester- or L-ribose riboic acid-cleaving activity] after the 15th round selection in a test tube -8 and 11-17). Boxes show regions with a high degree of sequence similarity.

Figures 10A-10B show (Figure 10A) 2 ': 10-16 and (Figure 10B) L-15-30 DNA enzyme, the dependence of the catalytic rate on Mg2 + concentration. The curve represents the best curve based on the following equation: U cadaver moxibustion and [Mg2 +] / ([Mg2 +] + L), where ‘is the presence of saturated Mg2 + ^, and ^ is the apparent cutting constant of Mg2 +. Figures 11A-11B show the temperature dependence of DNA-catalyzed reactions against (Figure 11A) 2 :, 10-16 and (Figure 11B) L: 15-30 DNA enzymes. Reaction conditions: 25 mM MgCl2, 150 mM NaCl and pH 7.5. Figures 12A-12B show the bivalent metal dependence of DNA-catalyzed reactions against (Figure 12A) 2 :: 10_16 and (Figure 12B) L: 15-30 DNA enzymes. Reaction Conditions · 10 mM M2 +, 150 mM NaCl, pH 7.5 and 37 ° C. The metal goes from 87851 -54- 200413528 to the right to reduce the pka value of the equivalent metal hydrate. Figures 13A-13D show the MALDI mass spectra of oligonucleotide products, starting from the catalytic reaction of 2s: 10-16 and L: 15-30 DNA enzymes. (Fig. 13A) From the product of the substrate containing 2 ', 5'-phosphodiester, the main product ion has an expected m / z of 3,346. (Fig. 13B) From the product containing 2', 5'-phosphodiester Substance 3, -product 'has 4,200 expected z / z; (figure from product containing L-ribonucleotides <5' of doublet, with expected m / z 2,971; (Figure 13D) -The 3, _ product of the ribonucleotide substrate with an expected m々 of 2,800. Figure 14 shows the uncatalyzed cleavage of the substrate used in this study. (Filled circle) contains 2 ', The substrate of 5'-phosphodiester has a moxibustion value of 1 × 10-6 minutes- ^; (open circle) is a substrate containing 3 ', 5'-phosphate diester, which has 2 &gt; &lt; 1. The value of -6 minutes' (through the square) contains a l · ribose riboic acid substrate, with a 10 minute h⑽ (open square) containing a 0-ribose riboic acid substrate, with 4 × 1〇- The value of 6 minutes · 1. The cutting rate can be obtained from the plot of the data as a function of time &lt; the optimal cutting line. Reaction conditions: 25 mM MgC12, 150 mM NaCl, pH 7.5 and 37 ° C. 15A-15B are diagrams according to the present invention. Illustration of chemical DNA molecules (nucleic acid sequences below) (SEQ ID NOS: 34, 29), aligned with the corresponding substrate (nucleic acid sequences above). In the two figures, the arrow indicates the cutting position, and the region 1 And 20 represents the acceptor nucleic acid sequence region complementary to the upstream (25) and downstream (5) flanking regions of the catalytic DNA molecule of the present invention. Fig. 15A illustrates a catalytic DNA molecule that can cleave a 2 ', 5'-linked mitochondrial ribonucleotide. Figure 15B illustrates a catalytic DNA molecule that can cleave a 3 ', 5'-linked acceptor L-enantiomer ribonucleotide. Figure 16 shows the DzyNA-PCR strategy used for homogeneous amplification reaction and detection of special nucleic acid sequences. 87851 -55- 200413528 Preface, j table &lt; 110 &gt; THE SCRIPPS RESEARCH INSTITUTE JOYCE, Gerald F. ORDOUKHANIAN, Phillip T. &lt; 120 &gt; RNA enzymes that cleave RNA with altered regions or enantioselectivity

&lt; 130 &gt; SCRIP1550TW &lt; 140 &gt; 092125352 &lt; 141 &gt; 2003-09-15 &lt; 150 &gt; US 60 / 410,973 &lt; 151 &gt; 2002-09-16 &lt; 160 &gt; 34 &lt; 170> Patentln version 3.1 &lt; 210 &gt; 1 &lt; 211 &gt; 50 &lt; 212 &gt; DNA &lt; 213> Artificial sequence &lt; 220 &gt; &lt; 223> Catalytic DNA fragment &lt; 400 &gt; 1 gggaccggcc actcggaggc atccatcgtt gcagaccttc 50 ltccccctgc 50 &lt; 210 &gt; 2 &lt211; 50 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; catalytic DNA fragment &lt; 400 &gt; 2 gggaccggcc actcggaggc atccatcgtt gcaaaccttg ttccccctgc 50 &lt; 210 &gt; 3 &lt; 211 &gt; 50 &lt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Catalytic DNA Fragment &lt; 400 &gt; 3 gggaccggcc actcggaggc atccatcgtt gcagacctcc ttccccctgc 50 &lt; 210 &gt; 4 &lt; 211 &gt; 50 &lt; 212> DNA &lt; 213 &gt; Sequence &lt; 220 &gt; &lt; 223> catalytic DNA fragment 87851 200413528 &lt; 400 &gt; 4 gggaccggcc actcggaggc atccatcgtt gcagaccttc ttccccctgc 50 &lt; 210> 5 &lt; 211> 50 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; catalytic DNA fragment &lt; 220 &gt; &lt; 221 &gt; misc feature &lt; 222 &gt; (3〇) .T (30) &lt; 223 &gt; n is any nucleic acid

Λ Λ Λ Λ 0 12 3 2 2 2 2 2 2 2 2 &lt; VVV misc-feature (33) di (33) η is any nuclear acid &lt; 400 &gt; 5 gggaccggcc actcggaggc atctatcgtn gcngaccttc ttccccctgc 50 &lt; 210> 6 &lt; 211 &gt; 50 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; catalytic DNA fragment Λ Λ Λ Λ 0 12 3 2 2 2 2 2 2 2 2 V &lt; &lt; &lt; misc-feature (43) .: (43) n is any nuclear fenamic acid &lt; 400 &gt; 6 gggaccggct actcggagtg cttcgtatgt cggtgagggt ctncctcccc 50 &lt; 210 &gt; 7 &lt; 211 &gt; 50 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &lt; 223 &gt; Catalytic DNA fragment &lt; 220 &gt; &lt; 221> misc feature &lt; 222 &gt; (24)-(49) &lt; 223 &gt; n is any island nucleic acid &lt; 400 &gt; 7 87851 200413528 gggaccggcc actcggaggc atcnatngtt gnggaccttt ttccccccnc column order NAA 850DN people &lt; 210 &gt; &lt; 211 &gt; &lt; 212 &gt; &lt; 213 &gt; &lt; 220 &gt; &lt; 223> Catalytic DNA fragment &lt; gag &gt; gcctgt actct gcc gcagaccttc ttccccctgc &lt; 210〉 9 &lt; 211〉 41 &lt; 212〉 DNA &lt; 213 &gt; Sequence &lt; 220 &gt; &lt; 223 &gt; catalytic DNA &lt; 400 &gt; 9 gcgagagtgg tttagggacc ggcactcgga gtgcagagag g &lt; 210 &gt; 10 &lt; 211〉 37 &lt; 212〉 DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223> Catalytic DNA &lt; 400> 10 gcgagagtgg ggaccggcca ctcggagtgc agagagg &lt; 210 &gt; 11 &lt; 211> 48 &lt; 212> DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &lt; 223 &gt; Catalytic DNA fragment &lt; 220 &gt; &lt; 221 &gt; misc-feature &lt; 222 &gt; (20) · (37) &lt; 223〉 n is any nucleic acid &lt; 400 &gt; 11 ggcgatcgtc tcagacatgn atnncatctt ggagggncag ttcgtcca &lt; 210 &gt; 12 &lt; 211> 50 &lt; 212 &gt; DNA &lt; 213> Artificial sequence 87851 200413528 &lt; 220 &gt; &lt; 223 &gt; Catalytic DNA fragment &lt; 400 &gt; 12 gaccggtcgc cttagacttg cagagtcgat gacgctcgta tcccactggg 50 &lt; 210 &gt; 13 &lt; 211 &gt; 50 DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &lt; 223 &gt; Catalytic DNA fragment &lt; 400 &gt; 13 gcacgatcgt cttagacatg ctgaggtctt gctctctaca gttgccgtca 50 &lt; 210 &gt; 14 &lt; 211 &gt; 50 &lt; 212 &g; DNA &lt; 213 t; artificial sequence &lt; 220 &gt; &lt; 223 &gt; catalytic DNA fragment &lt; 400 &gt; 14 gctgatcgtc ccagacatgc atagtccaac tctccctgac acccttagca 50 &lt; 210 &gt; 15 &lt; 211> 51 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; Catalytic DNA fragment &lt; 400 &gt; 15 &lt; 210 &gt; &lt; 211 &gt; &lt; 212〉 &lt; 213〉 gacgatcgtc ttagacatgc acgttatcgc gacgcctcga acggtccgcc c 51 6 o 1 DNA artificial sequence &lt; 220 &gt; 223 &gt; Catalytic DNA fragment &lt; 400 &gt; 16 gacgatcgtc tcagacataa atccgttagt ctctgttgtt ttgcgcgcta 50 &lt; 210〉 &lt; 211〉 &lt; 212〉 &lt; 213〉 7 o 1 5 DNA artificial sequence 87851 -4- 200413528 &lt; 220 &gt; &lt; 220 &gt;; 223> catalytic DNA fragment &lt; 400 &gt; 17 gacgagggtc ttggacataa atcggacgtc gatgtgacag caccagtccc 50 &lt; 210 &gt; 18 &lt; 211> 29 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; catalytic &lt; 400 &gt; 18 gcctcctcat cgtcttagac agcctctcc 29 &lt; 210 &gt; 19 &lt; 211〉 5 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223> Upstream'-Loop Fragment &lt; 400 &gt; 19 gttta 5 &lt; 210 &gt; 20 &lt; 211 &gt; 5 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220> &lt; 223 &gt; upstream loop fragment &lt; 400 &gt; 20 gctta 5 &lt; 210 &gt; 21 &lt;; 211 &gt; 4 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; upstream loop band fragment &lt; 400 &gt; 21 gtta 4 &lt; 210 &gt; 22 &lt; 211 &gt; 10 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence 87851 &lt; 220 &gt; 200413528 &lt; 223 &gt; downstream loop &lt; 400 &gt; 22 ccactcggag &lt; 210> 23 &lt; 211 &gt; 42 &lt; 212> DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; Primers &lt; 220 &gt; &lt; 221 &gt; misc_feature &lt; 222 &gt; (23). ~ (23)

&lt; 223> n is 2 ', 5'-rG or L-rA &lt; 400 &gt; 23 ttttagagac gatgacgatg cantcggaca gtcgcgagac tg &lt; 210 &gt; 24 &lt; 211 &gt; 84 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &lt; 223 &gt; template sequence

&gt; &gt;> ccga &lt; 210 &gt; 25 &lt; 211〉 19 &lt; 212 &gt; DNA &lt; 213〉 artificial sequence &lt; 220 &gt; &lt; 223〉 primer &lt; 400 &gt; 25 tcggacagtc gcgagactg &lt; 210> 26 &lt; 211 &gt; 27 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence 87851 -6-200413528 &lt; 220 &gt; &lt; 223〉 Primer &lt; 220 &gt; &lt; 221 &gt; misc-feature &lt; 222〉 (10). :( 12) &lt; 223 &gt; η is a non-basic nucleotide analog &lt; 400 &gt; 26 aacaacaacn nngtgccaag cttaccg 27 &lt; 210〉 27 &lt; 211> 15 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &lt; 223 &gt; Primer &lt;; 400 &gt; 27 gtgccaagct taccg 15 &lt; 210 &gt; 28 a &lt; 211 &gt; 11 &lt; 212〉 DNA &lt; 213> artificial sequence &lt; 220 &gt; &lt; 223 &gt; downstream region of catalytic functional site &gt; &gt; &gt; &gt; 0 12 3 2 2 2 2 2 2 2 2 V &lt; V &lt; misc a feature (1). 0), ^ n are the required cytosine nuclear residues

&gt; &gt; &gt; &gt; 0 12 3 2 2 2 2 2 2 2 2 V V V V misc-feature (2) .. (2) n is a cytosine or thymidine residue

&gt; &gt; &gt; &gt; 0 12 3 2 2 2 2 2 2 2 2 V &lt; VV nisc -feature 11). One (11) ^ Complementary Nucleic Acids Binding to the Nucleic Acid Nucleic Acid Sequence # 酸 &lt; 400 &gt; 28 nnactcggag η 11 &lt; 210 &gt; 29 &lt; 211> 14 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence 87851 200413528 &lt; 220 &gt; &lt; 223 &gt; a confirmation functional site and a catalytic functional site &lt; 220 &gt; &lt; 221 &gt; misc-feature &lt; 222 &gt; (!) .. 〇) &lt; 223 &gt; Complementary nucleic acid binding to the acceptor nucleic acid sequence, located immediately downstream of the cleavage position on the acceptor nucleic acid sequence &lt; 220 &gt; &lt; 22 1 &gt; misc_feature &lt; 222 &gt; (2) .. (2) &lt; 223 &gt; η is a nuclear thymocyte or guanidine residue &lt; 220 &gt; &lt; 22 1 &gt; misc-feature &lt; 222 &gt; (3) · [3) &lt; 223 &gt; η is a dense lake or fox residue

Λ Λ Λ &gt; 0 12 3 2 2 2 2 2 2 2 2 &lt; &lt; VV misc-feature (5) .. [5) η is a cytosine or thymidine residue Λ &gt; Λ Λ 0 12 3 2 2 2 2 2 2 2 2 VVV &lt; misc-feature (7): Γ7) η is a cytosine or thymidine residue &lt; 220 &gt; &lt; 221〉 misc-feature &lt; 222 &gt; (8) .. (8) &lt; 223> η is a cytosine or thymidine residue Λ Λ Λ Λ 0 12 3 2 2 2 2 2 2 2 2 &lt; VV &lt; misc__feature (9) .. (9) n is adenine nucleoside or guanidine residue &lt; 220 &gt; &lt; 221> misc_feature &lt; 222 &gt; (13) &quot; (13) &lt; 223 &gt; η adenine nuclear thymidine or thymidine Residues &lt; 220 &gt; &lt; 221 &gt; misc-feature &lt; 222 &gt; (14): :( 14) ... ^ &lt; 223 &gt; Upstream two nucleotides &lt; 400 &gt; 29 87851 200413528 nnngncnnng acnn &lt; 210 &gt; 30 &lt; 211〉 12 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &lt; 223> Catalytic DNA fragment &lt; 400 &gt; 30 tcgtcttaga ca &lt; 210> 31 &lt; 211 &gt; 20 &lt; 212 &gt; DNA &lt; 213 & g t; artificial sequence &lt; 220 &gt; &lt; 223 &gt; catalytic DNA fragment &lt; 220 &gt; a &lt; 221〉 misc a feature &lt; 222 &gt; (1) .. (1)

&lt; 223 &gt; n is -rG or L-rA &lt; 400 &gt; 31 ntcggacagt cgcgagactg &lt; 210 &gt; 32 &lt; 211〉 25 &lt; 212 &gt; DNA &lt; 213> artificial sequence &lt; 220 &gt; &lt; 223> target sequence &lt; 400 &gt; 32 cctctctgca gtcggacact ctcgc &lt; 210 &gt; 33 &lt; 211> 18 &lt; 212 &gt; DNA &lt; 213> artificial sequence &lt; 220 &gt; &lt; 223 &gt; target sequence &lt; 400 &gt; 33 ggagaggcat gaggaggc &lt; 210 &gt;; 34 &lt; 211 &gt; 21 &lt; 212 &gt; DNA &lt; 213> artificial sequence 87851 200413528 &lt; 220 &gt; &lt; 223 &gt; catalytic DNA fragment

Λ Λ Λ &gt; ο, —, 12 3 2 2 2 2 2 2 2 2 VVVV misc-feature (1) · (2) η is a well-known nuclear fenamic acid &lt; 220 &gt; &lt; 221 &gt; misc-feature &lt; 222 &gt; (7) .. ^ 7) &lt; 223 &gt; η is the V nuclear acid in the central stem region &lt; 220 &gt; &lt; 221 &gt; misc-feature &lt; 222> (8) · [8] &lt; 223 &gt; η is a cytosine nuclear residue &lt; 220 &gt; &lt; 221 &gt; misc-feature &lt; 222 &gt; (9). [9) &lt; 223 &gt; η is a guanidine residue

Λ Λ Λ Λ ο—- 2 3 2 2 2 2 2 2 2 2 V V V V misc a feature (10) :( 10) ri binds to the complementary nucleotide on the acceptor nucleic acid sequence, which is immediately downstream of the cutting position

Λ Λ Λ Λ 0 12 3 2 2 2 2 2 2 2 2 &lt; VVV misc one feature (11) two (11) η is the required cytosine nuclear tritium residue &lt; 220 &gt; &lt; 221 &gt; mi sc -Feature &lt; 222 &gt; (12) · T (12) &lt; 223 &gt; η is a pyrimidine or thymidine residue

<gt; &gt; Nucleic acid &lt; 400 &gt; 34 nngggannnn nnactcggag η 21 87851 10-

Claims (1)

200413528 Patent application park: 1. A catalytic single-stranded DNA molecule comprising one or more loop band regions and one or more binding regions, wherein the binding region binds to the complementary sequence of the acceptor nucleic acid sequence, and The catalytic DNA molecule has a site-specific endonuclease activity specific to the cleavage site in the acceptor nucleic acid sequence, and the cleavage site includes a non-naturally occurring single-stranded ribonucleic acid. 2. The catalytic DNA molecule according to item 1 of the scope of the patent application, wherein the non-naturally generated stranded nucleic acid comprises a 2 ', 5' linked residue. 3. The catalytic DNA molecule according to item 2 of the scope of the patent application, wherein the 2,5, linked residues are 2,5'-linked adenine nucleotides or guanine nucleotides. 4. The catalytic DNA molecule according to item 1 of the patent application, wherein the non-naturally generated single-stranded nucleic acid includes an L-enantiomeric residue. 5. The catalytic DNA molecule according to item 4 of the Shen Yan patent scope, wherein the L-enantiomeric residue is a 3 ', 5, _ linked adenine nucleotide residue. 6. The catalytic DNA molecule according to item 1 of the application, wherein the acceptor nucleic acid sequence is adhered to the catalytic DNA molecule. 7. The catalytic DNA molecule according to item 1 of the scope of patent application, wherein the endonuclease activity can be enhanced by the presence of Mg2 +. 8. The catalytic DNA according to item 1 of Shenyan's patent scope, wherein the endonuclease activity includes a hydrolytic cleavage of a phosphate bond at the cleavage position. 9. The catalytic DNA molecule according to item 1 of the patent application scope, wherein the Km value exhibited by the catalytic DNA molecule is less than about 1 μM. 10. Catalytic activity 1)] human molecule according to item 丨 of the scope of patent application, wherein the nucleic acid-87851 11200413528 endonuclease activity can be enhanced by the presence of divalent cations. The catalytic DNA molecule according to item 10 of the application, wherein the divalent cation is selected from the group consisting of Pb2 +, Mg2 +, Zna and Ca2 '. 12. 13. 14. The catalytic single-stranded DNA molecule according to item 7 of the scope of the patent application, which comprises: a)-a catalytic functional site, including: i) a downstream region that can form an annular zone, which includes the heart XiX2ACTCGGA GX3_3 ( (SEQ ID NO: 28), meaning is the cytosine nuclear residue, as needed, &amp; is a cytosine nuclear thymidine or thymidine residue, and &amp; binds to a complementary nucleus on the acceptor nucleic acid sequence Nucleic acid, two nucleotides upstream from the cleavage position; 11) A central stem region, including 5LZiZ2Z3Z4_3, immediately to the downstream loop 5 '' where Z4 binds to a complementary nucleic acid sequence of a complementary nucleic acid ' It is immediately downstream of the cutting position; and iii) an upstream zone that can form an endless zone, including 4 nucleotides 5 'to the inner dry zone, where the upstream zone contains 5, _GGga_3, and b) — Confirm the functional parts, including an upstream side connection area and a downstream side connection area. The 'upstream side connection area' is immediately connected to 5 of the upstream endless belt, and the downstream side connection area is immediately connected to 3 of the downstream endless belt. The catalytic DNA molecule according to item 12 of the application, wherein Z2 is a nucleoside residue and Zs is a guanidine residue. The catalytic DNA molecule according to item 12 of the application, wherein the downstream band contains 5, -CCACTCGGAG-3, (SEQ ID NO: 22). 87851 -2-200413528 15. The catalytic DNA molecule according to item 12 of the scope of patent application, wherein Z4 is a guanidine residue, X1 is a cytoplasmic nucleus residue, X2 is a cytoplasmic nucleus residue, and Xs Is a pulse residue. 16. The catalytic DNA molecule according to item 12 of the application, wherein the upstream loop contains 5f-YGGGA-3 ', where Y is 0 to 5 nucleotides. 17. A catalytic DNA molecule according to item 16 of the patent application, wherein Y contains 5, -TTA-3 ,. 18. A catalytic DNA molecule according to item 17 of the patent application, wherein Y comprises 5, -GTTTA-3, (SEQ ID NO: 19). 19. A catalytic DNA molecule according to item 17 of the patent application, wherein Y comprises 5, -GCTTA-3 '(SEQ ID NO: 20). 20. The catalytic DNA molecule according to item 17 of the application, wherein Υ includes 5, -GTTA-3, (SEQ ID NO: 21). 21. The catalytic DNA molecule according to item 12 of the application, wherein the catalytic DNA molecule includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. 22. The catalytic single-stranded DNA molecule according to item 9 of the patent application scope, comprising: a) a catalytic functional site capable of forming a loop, which includes S'-XiXdsGXztCXsXsXTGACXsXrS '(SEQ ID NO: 29), where Xi A complementary nucleotide that binds to the acceptor nucleic acid sequence, immediately downstream of the cleavage site, χ2 is a thymidine or guanidine residue, X3 is a cytosine residue or a guanidine residue, and X4 is a cytosine residue Or thymidine residue 87851 200413528 group, X5 is a cytosine core or a thymidine residue, the heart is a cytosine core or a thymidine residue, x? Is an adenine nucleoside or a guanidine residue, X8 is an adenine or thymidine residue, and &amp; binds to a complementary nucleotide on the acceptor nucleic acid sequence, two nucleotides upstream of the cleavage position; and b)-confirm the functional site, It includes an upstream side connection area and a downstream side connection area. The upstream side connection area is immediately adjacent to the catalytic function portion 5, and the downstream side connection area is immediately adjacent to the catalytic function portion 3. 23. The catalytic DNA molecule according to item 22 of the scope of application, wherein 乂 2 is a cytosine residue, X3 is a cytosine residue, χ4 is a thymine depletion 甞 residue, and X5 is a thymine For nucleocapsid residues, χ6 is a thymine nuclear oath residue, X7 is an adenine nucleoside residue, and χ8 is an adenine nucleoside residue. 24. The catalytic DNA molecule according to item 22 of the application, wherein the loop comprises 5'-TCGTCTTAGCA-3, (SEQ ID NO: 30). 25. The catalytic DNA molecule according to item 22 of the application, wherein the catalytic DNA molecule includes: SEQ ID NO: 11, SEQ ID NO: 12, SEQ_ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID, NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. 26. The catalytic DNA molecule according to item 22 of the application, wherein the catalytic DNA molecule includes SEQ ID NO: 18. 27. A method for detecting a target nucleic acid sequence, the method comprising: a) blending in an amplification reaction buffer solution: i) a nucleic acid sample; i0-polymerase; 87851 200413528 non-naturally occurring single strands of lobster Nucleic acid sequence, including an interactive tag pair consisting of a first tag and a second tag, which are directly or indirectly attached to oligonucleotides, where the first tag is cut by a non-naturally occurring ribonucleotide Separate from the second flag, · IV) — forward primer that can bind to the first strand of the target nucleic acid sequence (3 copies and instruct the target nucleic acid sequence to synthesize a polynucleotide in the forward direction; and V) — reverse Primer, which can be bound to the first 3 &lt; region of the target nucleic acid sequence, and instructs the target nucleic acid sequence to synthesize hydroglyceric acid in the reverse direction, and contains a catalytic single-stranded DNA molecule according to the scope of the patent application Complementary, in which the catalytic single-stranded DNA molecule can cleave the acceptor nucleic acid sequence at the cleavage position; b) cultivate the connected group # under the expansion reaction conditions to provide the expansion of the target nucleic acid sequence and synthesize the catalytic property Strands of DNA molecules, and cleavage of the acceptor nucleic acid sequence by the catalytic material strands of DNA molecules, so as to release the interaction between the-flag and the brother flag; and e) detection of the-flag can be detected Targeted nucleic acid sequence. 28. 29. 30. 31. According to the ancient dentist in item 27 of the scope of the patent application, the non-naturally occurring ribose ribonucleic acid cleavage site includes a 2, 5, · -linked residue. According to the 27th item of the scope of the patent application, Tugan + u ^, Anibei Wanwan method, where the non-naturally occurring ribose ribonucleotide cleavage position contains L-enantiomeric residues. According to Gan + ^, Yi Wan Fa, item 27 of the scope of patent application, the first flag is the fluorescent part, and the second flag is the refrigerant. According to item 27 of the scope of patent application "&lt; Wan Qu, among which catalytic single-stranded DNA 87851 200413528 molecule 疋 catalytic single-strand DNA molecule according to the scope of patent application item 1. 32. Method according to the scope of patent application item 31 The catalytic single-stranded DNA molecule is based on the catalytic single-stranded 1)] ^ molecule in accordance with the scope of the patent application No. 6 33. The method according to the scope of the patent application No. 28, wherein the 2, 5, 5, link 2,5-linked adenine nucleotide or guanine nucleotide ribonucleoside acid residue. 34. The method according to item 33 of Shenyan patent scope, wherein the catalytic single-stranded molecule is based on Catalytic single-stranded DNA molecule in the scope of application for patent item 1. 35. The method according to the scope of the patent application in item 33, wherein the catalytic single-stranded DNA molecule is based on the catalytic single-stranded DNA molecule in item 3 of the applied patent scope. 3 6. The method according to item 27 of the patent application, wherein the non-naturally occurring single-stranded nucleic acid includes L-enantiomeric residues. 37. The method according to item 36 of the patent application, wherein the L-enantiomer residue Base is 3 ', 5 ^ linked adenine Taste acid ribose riboic acid residues. 3 8. The method according to item 37 of the scope of patent application, wherein the catalytic single-stranded DNA molecule is the catalytic DNA molecule according to item 1 of the scope of patent application. 39. According to the scope of patent application The method of item 27, wherein the polymerase is a Thermusaquaticus DNA polymerase, and wherein the amplification reaction is performed by using a polymerase chain reaction. 40 · —a non-naturally occurring single-stranded nucleic acid substrate, Includes an interactive tag pair consisting of a first tag and a second tag, which are directly or indirectly attached to the oligonucleotide, where the first tag and the second tag consist of 2, 5 ,,- Separated by the cleavage position of linked residues or L-enantiomeric residues. 41 · According to the acceptance of item 40 in the scope of the patent application, the first weave is a 87851 200413528 fluorescent part, and the second This flag is a refrigerant. 42. 43. 44. 45. 46. 47. According to the subject matter of the scope of the patent application, the cutting position includes 2, 5, 5, residues connected. According to the scope of the patent application, 42 Acceptance of item, where the cutting position includes 2 5'-linked adenine riboic acid or guanine nucleotide ribonucleotide residues. According to the subject matter in the scope of patent application No. 43, wherein the cleavage position includes l_ enantiomeric residues. Substances of item 44 wherein the L-enantiomeric residue is a 3 ', 5'-linked adenine ribonucleotide ribonucleotide residue. One contains two or more of the catalytic properties according to item 1 of the scope of the patent application A composition of DNA molecule groups, in which each group of catalytic ONA molecules can cleave a nucleic acid sequence that is different in quality. A method for identifying catalytic DNA molecules having position-specific endonuclease activity and specificity for non-naturally occurring ribonucleic acid cleavage positions. This method includes: a) building a double-stranded nucleic acid molecule library, It contains a non-naturally occurring ribonucleoside cut site 'and includes nucleotide regions of any sequence that may interact with the cleavage site region; b) captures one strand of a double-stranded nucleic acid molecular library, which can provide captured A single-stranded nucleic acid molecule library; c) growing the captured single-stranded nucleic acid molecule library under cleavage conditions, causing cleavage at the cleavage site and releasing the cleavage nucleic acid molecule; and d) separating the cleavage nucleic acid molecule, so that A catalytic DNA molecule with a position _ 87851 200413528 specific endonuclease activity was identified, which is specific for non-naturally occurring ribonucleotide cleavage positions. μ 48. The method according to item 47 of the scope of patent application, wherein the method includes: ^ Θ enlarges the cleaved nucleic acid molecule and repeats steps “1 to 4 times; g) randomly selects the selectively enlarged cleaved nucleic acid molecule Mutation to form a mutated and cleaved nucleic acid molecule; and h) expanding the mutated and cleaved nucleic acid molecule and repeating steps b_e 丨 to 20 times. 49. The method according to item 48 of the scope of the claimed patent, wherein in subsequent steps The cleavage conditions are changed during repeated sentences, so the cleavage reaction must be more efficient so that nucleic acid cleavage can occur. 50. According to the method in the 47th scope of the patent application, the non-naturally occurring ribonucleic acid cleavage position is 2, 5, -linked guanine ribonucleoside ribonucleoside cleavage site. 5 1. The method according to item 47 of the patent application scope, wherein the non-naturally occurring ribose nucleoside cleavage site is L-adenosine Acid ribonucleotide cleavage sites. — 52. — A set of nucleic acid sequences for detection of targets, this set includes ... A) — a single-stranded nucleic acid sequence that is unnaturally generated, Includes a pair of interactively active flags consisting of a first flag and a second flag, which are directly or indirectly attached to the oligonucleotide, where the first flag and the second flag are non-naturally generated ribose nuclei Separate by the cleavage position of acetic acid; B) — a forward primer that can bind to the first strand of the target nucleic acid sequence 3, 87851 -8-200413528 Shao Fen 'and instruct the target nucleic acid sequence to synthesize polynuclear acid in the forward direction, And C) a reverse primer that contains a region that can bind to the second strand of the target nucleic acid sequence, and instructs the target nucleic acid sequence to synthesize polynucleotides in the reverse direction 'and includes the first The catalytic single-stranded DNA of item is a complement of a daughter, wherein the catalytic single-stranded DNA molecule can cleave the acceptor nucleic acid sequence at the position of the cutting damage. 53. 54. 55. 56. 57. 58. 59. 60. 61. According to the set of patent application scope 52, the non-naturally occurring ribonucleoside cleavage position contains 2 ', 5f linked residues. According to the set of patent application scope 52, the non-naturally occurring The ribonucleotide cleavage site contains the L-enantiomer According to the set of 52 of the scope of patent application, the first standard weave is a fluorescent β f knife and the first standard is a quenching agent. According to the set of 52 scope of the patent application, the catalytic property is The single-stranded DNA molecule is a catalytic single-stranded DNa molecule according to item 1 of the scope of the patent application. According to the 56th set of patent scope of the patent, the catalytic single-stranded DNA knife Single-stranded DNA molecule. The set according to item 53 of the patent application, wherein the 2,5, linked residues are 2 ', 5'-linked adenine ribonucleotide or guanine nucleotide ribose Nucleotide residues. The kit according to item 58 of the scope of patent application, wherein the catalytic single-stranded DNA molecule is the catalytic single-strand DNA molecule according to item 1 of the scope of patent application. The kit according to item 58 of the scope of patent application, wherein the catalytic single-stranded DNA knife is based on the catalytic single-strand DNA molecule of item 3 of the scope of patent application. According to the set of patent application scope 52, the non-naturally occurring single 87851 200413528 strand nucleic acid contains L-enantiomeric residues. 62. The set according to the scope of patent application No. 61, wherein the L-enantiomeric residue is a linked adenine ribonate ribonucleoside residue. 10- 87851