JP2006513701A5 - - Google Patents

Claims (198)

a) a first subject sequence having about 3 to 150 nucleotides each independently and substantially complementary to each other to form a first hybridized duplex; and A first nucleic acid sequence pair consisting of one complementary sequence;
b) at least one recognition element joined to at least one of the first target sequence and the first complementary sequence and specifically interacting with at least one target factor; and c) optionally, At least one, preferably all, of the components comprising: at least one detectable label that generates a characteristic signal whose magnitude is indicated as a function of the amount of said first hybridized duplex A probe included in a state connected to
When the target factor is present, the amount of the first hybridized duplex due to the interaction between the target factor and the recognition element changes compared to the amount when the target factor is not present. A probe whose characteristic signal is changed by The probe further comprises a second nucleic acid sequence pair that is a competing nucleic acid sequence consisting of a second target sequence and a second complementary sequence,
The second target sequence and the second complementary sequence each independently have about 3 to 150 nucleotides and are substantially complementary to each other, so that the second hybridized duplex (second hybridized duplex) );
The first target sequence and the second target sequence have an overlapping region consisting of at least one nucleotide while being included in one target sequence,
Due to the interaction between the target factor and the recognition element when the target factor is present, the first hybridized duplex and the second hybridized duplex are compared to when the target factor is absent. The probe according to claim 1, wherein the characteristic signal is changed by decreasing the amount of any one of the heavy chains and increasing the amount of the remaining one.   The probe according to claim 2, wherein the recognition element is joined to the first target sequence excluding the overlapping region or the first complementary sequence. The target sequence and the complementary sequence are selected from the group consisting of DNA, a derivative of DNA, RNA, PNA, LNA, and a mixture of at least two of DNA, a derivative of DNA, RNA, PNA, and LNA. The probe according to any one of 2 and 2.   The affinity probe according to claim 1, wherein the recognition element is a probe ligand, and the target factor is a receptor factor that can specifically bind to the probe ligand.   The affinity probe according to claim 5, wherein the melting temperature of the first hybridized duplex is decreased by at least 1 ° C. when the receptor factor binds to the probe ligand.   The affinity probe according to claim 5, wherein the probe ligand is covalently linked to the first target sequence or the first complementary sequence by at least one linking element.   The linking element is selected from the group consisting of a chemical bond, a divalent atom, a divalent chemical moiety, and a multivalent chemical moiety. 8. The affinity probe according to 7. The probe ligand is a chemical ligand, a biochemical ligand, an antigen, an antibody, an antibody fragment, an enzyme, an enzyme substrate, an enzyme inhibitor, a hormone, an antibiotic. , Narcotic, toxin, polypeptide, protein, protein fragment, targeting sequence, transit peptide, glycoprotein, lipid, phospholipid ( 6. The affinity probe according to claim 5, selected from the group consisting of phospholipids, polysaccharides, carbohydrates, nucleic acids, and peptide nucleic acids.   The recognition element is a probe substrate including a destabilizing factor and at least one cleavage site, and the target factor is a reaction inducer capable of specifically cleaving the cleavage site, and the first end of the cleavage site Is joined to the destabilizing factor and the second end of the cleavage site is covalently linked to the first target sequence or the first complementary sequence by at least one linking element. The cleavable probe as described.   The cleavable probe according to claim 10, wherein the melting temperature of the first hybridized duplex is increased by at least 1 ° C when the cleavage site is cleaved by the reaction inducer. The cleavable probe according to claim 10, wherein the linking element is selected from the group consisting of a chemical bond, a divalent atom, a divalent chemical moiety, and a multivalent chemical moiety.   The cleavable probe according to claim 10, wherein the destabilizing factor is covalently or non-covalently linked to the first end of the cleavage site by a linker.   The destabilizing factor is streptavidin or a derivative thereof that binds to biotin, and the biotin is covalently linked to the first end of the cleavage site by a linker. The cleavable probe as described.   14. The cleavable probe according to claim 13, wherein the linker is selected from the group consisting of a chemical bond, a divalent atom, a divalent chemical moiety, and a multivalent chemical moiety.   The cleavable probe according to claim 10, wherein the cleavage site is specifically cleaved by a protease, endonuclease, lipase, or glycosidase.   The cleaving probe according to claim 16, wherein the cleavage site is specifically cleaved by a mammalian or viral protease.   The cleaving probe according to claim 16, wherein the cleavage site is specifically cleaved by a protease associated with a human pathogen. The protease is cytomegalovirus (CMV), herpes simplex virus (HSV), hepatitis virus, plasmodium, human immunodeficiency virus (HIV), Kaposi 19. The cleavage property according to claim 18 , which is a protease expressed from sarcoma-associated herpesvirus (Kaposi's sarcoma-associated herpes virus: KSHV), yellow fever virus, flavivirus, or rhinovirus. probe. 17. The cleavable probe according to claim 16 , wherein the protease is a serine-type, cysteine-type, or aspartate-type protease. 19. The cleavable probe according to claim 18 , wherein the human pathogen is a yeast, a bacterium, a fungi, a nematode, a virus, or a protozoa. The cleavage site, blood clotting (blood coagulation), apoptosis (apoptosis), Alzheimer's disease (Alzheimer's disease), or extracellular matrix (extracellular matrix) and claims is specifically cleaved by a protease of a mammal associated with 17 The cleavable probe according to 1.   The recognition element is a probe substrate including at least one reaction site covalently linked to the first target sequence or the first complementary sequence by at least one linking element, and the target factor is destabilized The type I connectivity probe according to claim 1, which is a reaction inducer capable of specifically conjugating a factor to the reaction site. 24. The type I connectivity probe according to claim 23 , wherein the melting temperature of the first hybridized duplex is decreased by at least 1 ° C. when the destabilizing factor is joined by the reaction inducing factor. The linking element is selected from the group consisting of a chemical bond, a divalent atom, a divalent chemical moiety, and a multivalent chemical moiety. The type I connectivity probe according to 23 . 24. Type I connectivity according to claim 23 , wherein the destabilizing factor is a protein or protein complex having at least one reactive group that can be covalently linked to the reaction site in the presence of the reaction-inducing factor. probe. 27. The type I linking probe according to claim 26 , wherein the destabilizing factor is streptavidin or a derivative thereof bound to modified biotin having at least one reactive group. 24. The type I connectivity probe according to claim 23 , wherein the reaction site is joined to the destabilizing factor by ligase. 29. The type I connectivity probe according to claim 28 , wherein the ligase is selected from the group consisting of polynucleotide ligase, aminoacyl tRNA ligase, and biotin protein ligase.   The recognition element is a probe substrate including at least one reaction site covalently linked to the first target sequence or the first complementary sequence by at least one linking element, and the target factor is the reaction site The type II connectivity probe according to claim 1, which is a reaction inducing factor capable of specifically converting to a conjugation site to which a destabilizing factor can be conjugated. The type II connectivity probe according to claim 30 , wherein the melting temperature of the first hybridized duplex is decreased by at least 1 ° C when the destabilizing factor is joined by the reaction inducing factor. The type II connectivity probe according to claim 30 , wherein the linking element is selected from the group consisting of a chemical bond, a divalent atom, a divalent chemical moiety, and a multivalent chemical moiety. The type II connectivity probe according to claim 30 , wherein the destabilizing factor is a protein or protein complex capable of specifically binding to the conjugation site. The type II protein according to claim 30 , wherein the destabilizing factor is a protein or protein complex having at least one reactive group capable of being covalently linked to the conjugation site spontaneously or in the presence of a linking reagent. Connectivity probe. 35. The type II linking probe according to claim 34 , wherein the destabilizing factor is streptavidin or a derivative thereof bound to modified biotin having at least one reactive group. The type II connectivity probe according to claim 30 , wherein the reaction site is specifically converted by transferase. 37. The type II connectivity probe according to claim 36 , wherein the transferase is selected from the group consisting of a kinase and an acetyl-CoA transferase. The kinases include c-AMP dependent protein kinase (PKA), casein kinase I (casein kinase I: CKI), casein kinase II (case II kinase CKII), glycogen synthase kinase 3 (glycogen) synthase kinase 3: GSK-3), cdc2 protein kinase, calmodulin-dependent protein kinase II (CaMKII), insulin receptor (INSR), mitogen-activated protein Kinase (mitogen-activated protein kinase: MAPK), cGMP-dependent protein kinase (cGPK), phosphorylase kinase (PhK), protein kinase C (protein kinase C: PKC), p34 cdc2 protein Kinase (p34 cdc2 protein kinase), meiosis-activated mye Basic protein kinase (meiosis-activated myelin basic protein kinase: p44 mpk), smooth muscle myosin light chain kinase, epidermal growth factor receptor kinase (EGF-RK) And a protein tyrosine kinase pp60c-src (PTK). The type II connectivity probe according to claim 37, which is selected from the group consisting of protein tyrosine kinase pp60c-src (PTK). The type II connectivity probe according to claim 30 , wherein the reaction site is a site specific to a kinase, and the junction site contains a phosphate group attached by the kinase. 40. The type II connectivity probe according to claim 39 , wherein the destabilizing factor is an antibody or a receptor that can specifically bind to the junction site having the phosphate group attached by the kinase. The phosphate group is a thiophosphate group, and the destabilizing factor is an iodoacetyl group or a thiol group that can be covalently linked to the junction site in the presence of a linking reagent. The type II linking probe according to claim 39 , which is streptavidin or a derivative thereof bound to a modified biotin having at least one reactive group such as The destabilizing factor is composed of a synthetic polymer or analog thereof containing a divalent cationic, tricataionic, or polycataionic metal ion. The type II of claim 39, which is a nanoparticle, microparticle, bead, or membrane that can specifically bind to the junction site having the phosphate group. Connectivity probe.   The recognition element is a probe substrate including at least one reaction site covalently linked to the first target sequence or the first complementary sequence by at least one linking element, and the target factor is destabilized The type II (-)-linked probe according to claim 1, which is a reaction inducing factor capable of specifically converting the reaction site to which a factor can be conjugated to a non-zygous site. 44. The type II (−) connectivity probe according to claim 43 , wherein the melting temperature of the first hybridized duplex is decreased by at least 1 ° C. when the destabilizing factor is joined. 44. The type II (−) connectivity probe according to claim 43 , wherein the linking element is selected from the group consisting of a chemical bond, a divalent atom, a divalent chemical moiety, and a polyvalent chemical moiety. 44. The type II (−) connectivity probe according to claim 43 , wherein the destabilizing factor is a protein or protein complex capable of specifically binding to the reaction site. 44. The II of claim 43 , wherein the destabilizing factor is a protein or protein complex having at least one reactive group that can be covalently linked to the reaction site either spontaneously or in the presence of a linking reagent. -) Type connectivity probe. The type II (-) linkage according to claim 47 , wherein the destabilizing factor is streptavidin or a derivative thereof bound to a modified biotin having at least one of the reactive groups. Sex probe. 44. The type II (−)-linked probe according to claim 43 , wherein the reaction site is a site having a phosphate group that can be specifically dephosphorylated by phosphatase. The phosphatase is a phosphoprotein phosphatase such as protein tyrosine phosphatase, dual specificity phosphatase and protein Ser / Thr phosphatase; phosphatidylinositol -3,4-biphosphate 4-phosphatase, phosphatidylinositol-3,4,5-triphosphate 3 -phosphatase), phospholipid phosphatase such as SH2 domain-containing inositol phosphatase (SHIP) and membrane-associated phospholipid phosphatase; polynucleotide 3'-phosphatase (po 50. Type II (-) ligation according to claim 49 selected from the group consisting of: polynucleotide phosphatase such as lynucleotide 3'-phosphatase and polynucleotide 5'-phosphatase Sex probe. 50. The type II (−) linking probe according to claim 49 , wherein the destabilizing factor is an antibody or a receptor capable of specifically binding to the reaction site having the phosphate group. The phosphate group is a thiophosphate group, and the destabilizing factor is an iodoacetyl group or a thiol group that can be covalently linked to the reaction site in the presence of a linking reagent. 50. The type II (−) linking probe according to claim 49 , which is streptavidin or a derivative thereof bound to modified biotin having at least one reactive group as described above. The destabilizing factor is composed of a synthetic polymer or analog thereof containing a divalent cationic, tricataionic, or polycataionic metal ion. particles (nanoparticle), microparticles (microparticle), a bead (bead), or membrane (membrane), II, according to claim 49 capable of binding specifically to the reaction site having a phosphoric acid group ( -) Type connectivity probe.   The affinity probe according to claim 2, wherein when the target factor is not present, the first hybridized duplex is preferentially formed as compared to the second hybridized duplex. 55. The affinity probe according to claim 54 , wherein the second hybridized duplex is preferentially formed as compared to the first hybridized duplex when the target factor is present in excess.   The cleaving probe according to claim 2, wherein the second hybridized duplex is preferentially formed as compared to the first hybridized duplex when the target factor is not present. 57. The cleavable probe according to claim 56 , wherein when the target factor is present in an excessive amount, the first hybridized duplex is preferentially formed as compared to the second hybridized duplex.   The type I connectivity probe according to claim 2, wherein when the target factor is not present, the first hybridized duplex is preferentially formed as compared to the second hybridized duplex. . 59. The type I connectivity of claim 58 , wherein the second hybridized duplex is preferentially formed over the first hybridized duplex when the target factor is present in excess. probe.   The type II connectivity probe according to claim 2, wherein when the target factor is not present, the first hybridized duplex is preferentially formed as compared to the second hybridized duplex. . 61. The type II connectivity of claim 60 , wherein the second hybridized duplex is preferentially formed over the first hybridized duplex when the target factor is present in excess. probe.   The type II (-) according to claim 2, wherein the second hybridized duplex is preferentially formed in comparison with the first hybridized duplex when the target factor is not present. Connectivity probe. 63. The II (-) of claim 62 , wherein the first hybridized duplex is preferentially formed relative to the second hybridized duplex when the target factor is present in excess. Type connectivity probe.   The probe according to claim 1, wherein the detectable label is a non-interactive label selected from the group consisting of a phosphor, a luminescent material, a radioisotope, an enzyme, an antibody, an antigen, and an electrochemical label. The probe is a bimolecular probe composed of a first molecule including the first target sequence and a second molecule including the first complementary sequence, wherein the at least one detectable label is the first and second labels. The probe according to claim 64 , which is covalently linked to at least one of the molecules. 66. The probe of claim 65 , wherein the first molecule is immobilized on a support and the at least one detectable label is covalently linked to the second molecule. 66. The probe of claim 65 , wherein the second molecule is immobilized on a support and the at least one detectable label is covalently linked to the first molecule.   The probe according to claim 1, wherein the detectable label is an intercalating dye capable of preferentially binding to a double-stranded nucleic acid.   The at least one detectable label comprises an interactive label pair comprising a first label portion joined to the first target sequence and a second label portion joined to the first complementary sequence. The probe of claim 1, wherein the first and second label moieties interact when the first hybridized duplex is formed. The interactive label pair is composed of a phosphor and a quencher, and the interaction of the interactive label pair causes a change in fluorescence of the probe, and the change in fluorescence results in a decrease in intensity, a decrease in lifetime, polarization 70. The probe of claim 69 , selected from the group consisting of: A bimolecular probe wherein the probe comprises a first molecule containing the first target sequence containing the first label portion and a second molecule containing the first complementary sequence containing the second label portion 70. The probe of claim 69 . The probe according to claim 71 , wherein the first or second molecule is immobilized on a support. 70. The probe according to claim 69 , wherein the probe is a unimolecular probe in which the first target sequence and the first complementary sequence are covalently linked by a loop portion. 74. The probe according to claim 73 , wherein the loop portion links either the 5 ′ or 3 ′ end of the first target sequence to either the 5 ′ or 3 ′ end of the first complementary sequence. 74. The probe of claim 73 , wherein the loop portion is a nucleic acid sequence having about 4-100 nucleotides. The probe according to claim 75 , wherein the first target sequence, the loop portion, and the first complementary sequence are sequentially covalently linked in the 5 'to 3' direction or the 3 'to 5' direction. The probe according to claim 73 , which is immobilized on a support.   The probe is a single molecule probe, wherein the first target sequence and the first complementary sequence are covalently linked by a loop portion, and the at least one detectable label is a phase of the single molecule probe. An interactive label pair comprising first and second label portions joined at different positions, wherein when the first hybridized duplex is formed, the first and second label portions are The probe according to claim 1 which acts. The interactive label pair is composed of a phosphor and a quencher, and the interaction of the interactive label pair causes a change in fluorescence of the probe, and the change in fluorescence results in a decrease in intensity, a decrease in lifetime, polarization 79. The probe according to claim 78 , wherein the probe is selected from the group consisting of: 79. The probe of claim 78 , wherein at least one of the first and second label portions is covalently linked to the loop portion. The probe according to claim 78 , which is immobilized on a support. The probe includes a 5 ′ arm sequence covalently linked to the 5 ′ end of the first target sequence, and a 3 ′ covalently linked to the 3 ′ end of the first target sequence. A probe further comprising a nucleic acid arm sequence pair consisting of an arm sequence (3'arm sequence),
When the first target sequence is not hybridized to the first complementary sequence, the arm sequence pair forms a stemd duplex having about 3 to 35 complementary base pairs. ;
The detectable label comprises an interactive label pair consisting of a first label portion joined to the 5 ′ arm sequence and a second label portion joined to the 3 ′ arm sequence, the stem duplex The probe of claim 1, wherein the first and second label portions interact when the is formed. The probe includes a 5 ′ arm sequence covalently linked to the 5 ′ end of the first complementary sequence and a 3 ′ covalently linked to the 3 ′ end of the first complementary sequence. A probe further comprising a nucleic acid arm sequence pair consisting of an arm sequence (3'arm sequence),
When the first target sequence is not hybridized to the first complementary sequence, the arm sequence pair forms a stem duplex having about 3 to 35 complementary base pairs. ;
The detectable label comprises an interactive label pair consisting of a first label portion joined to the 5 ′ arm sequence and a second label portion joined to the 3 ′ arm sequence, the stem duplex The probe of claim 1, wherein the first and second label portions interact when a chain is formed. The probe is covalently linked to a first 5 'arm sequence that is covalently linked to the 5' end of the first target sequence and a 3 'end of the first target sequence. A first arm sequence pair consisting of a first 3 'arm sequence and a second 5' arm sequence (second) covalently linked to the 5 'end of the first complementary sequence. 5 'arm sequence) and a second arm sequence pair consisting of a second 3' arm sequence covalently linked to the 3 'end of the first complementary sequence A probe further comprising a nucleic acid arm sequence pair of
When the first sequence of interest is not hybridized to the first complementary sequence, the first arm sequence pair is a first stem duplex having about 3 to 35 complementary base pairs (first stem duplex). the second arm sequence pair forms a second stemd duplex having about 3 to 35 complementary base pairs;
The detectable label comprises a first label pair comprising a first label portion joined to the first 5 ′ arm array and a second label portion joined to the first 3 ′ arm array; Two pairs of interactive labels comprising a third label portion joined to a second 5 'arm arrangement and a second label pair comprising a fourth label portion joined to the second 3' arm arrangement A pair comprising: the first label pair interacts when the first stem duplex is formed and the second label pair interacts when the second stem duplex is formed Item 2. The probe according to Item 1. The interactive label pair is composed of a phosphor and a quencher, and the interaction of the interactive label pair causes a change in fluorescence of the probe, and the change in fluorescence results in a decrease in intensity, a decrease in lifetime, polarization The probe according to any one of claims 82 to 84 , which is selected from the group consisting of a change in wavelength and a change in wavelength. The probe according to any one of claims 82 to 84 , wherein the probe is a bimolecular probe including a first molecule including the first target sequence and a second molecule including the first complementary sequence. The probe according to claim 86 , wherein the first or second molecule is immobilized on a support. The probe, to the second component claims 82 is a unimolecular probe which are covalently linked by a loop portion comprising a first component and at least the first complement sequence including at least the first target sequence 84 The probe according to any one of the above.   The probe according to claim 112, which is immobilized on a support.   The probe according to claim 2, wherein the detectable label is a non-interactive label selected from the group consisting of a phosphor, a luminescent material, a radioisotope, an enzyme, an antibody, an antigen, and an electrochemical label. The probe is a trimolecular probe comprising a first molecule including the target sequence, a second molecule including the first complementary sequence, and a third molecule including the second complementary sequence, and the at least one detectable 96. The probe of claim 90 , wherein a unique label is covalently linked to at least one of the first, second and third molecules. If the at least one detectable label is covalently linked to at least one of the second and third molecules, and if both molecules are labeled, each molecule has a different label. 92. The probe of claim 91 . The probe according to claim 92 , wherein the first molecule is immobilized on a support. 92. The probe of claim 91 , wherein either the second or third molecule is immobilized on a support and the at least one detectable label is covalently linked to the first molecule. The probe is a bimolecular probe composed of a first molecule including one of the first and second complementary sequences and the target sequence, and a second molecule including the remaining sequence, and the first molecule includes The two sequences included are covalently linked by a loop moiety, and the at least one detectable label is covalently linked to at least one of the first and second molecules. The probe according to claim 90 . 96. The probe of claim 95 , wherein the first molecule is immobilized on a support and the at least one detectable label is covalently linked to the second molecule. 96. The probe of claim 95 , wherein the second molecule is immobilized on a support and the at least one detectable label is covalently linked to the first molecule.   The probe according to claim 2, wherein the detectable label is an intercalating dye capable of preferentially binding to a double-stranded nucleic acid.   The at least one detectable label comprises an interactive label pair comprising a first label portion joined to the first target sequence and a second label portion joined to the first complementary sequence. The probe of claim 2, wherein the first and second label moieties interact when the first hybridized duplex is formed.   The at least one detectable label comprises an interactive label pair comprising a first label portion joined to the second target sequence and a second label portion joined to the second complementary sequence. The probe of claim 2, wherein the first and second label moieties interact when the second hybridized duplex is formed.   Wherein the at least one detectable label comprises two different pairs of interactive labels comprising a first label pair and a second label pair, wherein the first label pair comprises the first object A first label portion joined to a sequence and a second label portion joined to the first complementary sequence, wherein the second label pair is joined to the second target sequence And a fourth label portion joined to the second complementary sequence, the first and second label portions interact when the first hybridized duplex is formed, and the second The probe according to claim 2, wherein the third and fourth label portions interact when a hybridized duplex is formed. 102. The probe of claim 101 , wherein the first and third label portions are the same portion. The interactive label pair is composed of a phosphor and a quencher, and the interaction of the interactive label pair causes a change in the fluorescence of the probe, and the change in fluorescence is caused by a decrease in intensity, a decrease in lifetime, polarization 102. The probe according to any one of claims 99 to 101 , which is selected from the group consisting of a change and a change in wavelength. 102. The probe according to claim 99 , wherein the probe is a trimolecular probe including a first molecule including the target sequence, a second molecule including the first complementary sequence, and a third molecule including the second complementary sequence. The probe according to one item. 105. The probe of claim 104 , wherein one of the first, second and third molecules is immobilized on a support. The probe is a bimolecular probe comprising a first molecule including two of the target sequence, the first complementary sequence and the second complementary sequence, and a second molecule including the remaining sequence, 102. The probe according to any one of claims 99 to 101 , wherein the two sequences contained in one molecule are covalently linked by a loop portion. 107. The probe according to claim 106 , wherein the first or second molecule is immobilized on a support. 102. The probe according to claim 99 , wherein the target sequence, the first complementary sequence, and the second complementary sequence are covalently linked by at least one loop portion. The probe according to 1. 109. The probe of claim 108 , wherein the loop portion is a nucleic acid sequence having about 4-100 nucleotides. The first complementary sequence, the first loop portion, the target sequence, the second loop portion, and the second complementary sequence are sequentially covalently linked in the 5 ′ to 3 ′ direction or the 3 ′ to 5 ′ direction. 110. The probe of claim 109 . The target sequence, the first loop portion, the first complementary sequence, the second loop portion, and the second complementary sequence are sequentially linked by a covalent bond in the 5 ′ to 3 ′ direction or the 3 ′ to 5 ′ direction. 110. The probe of claim 109 . The target sequence, the first loop portion, the second complementary sequence, the second loop portion, and the first complementary sequence are sequentially linked by a covalent bond in the 5 ′ to 3 ′ direction or the 3 ′ to 5 ′ direction. 110. The probe of claim 109 . The probe according to claim 108 , which is immobilized on a support.   The probe is a unimolecular probe, wherein the target sequence, the first complementary sequence, and the second complementary sequence are covalently linked by at least one loop portion, and the at least one detectable label Comprises at least one interactive label pair, each label pair comprising first and second label portions joined at different positions of the unimolecular probe, wherein the first or second hybridized two The probe of claim 2, wherein the first and second label moieties interact when a heavy chain is formed. 115. The probe of claim 114 , wherein at least one of the first and second label portions is covalently linked to the loop portion. The interactive label pair includes a phosphor and a quencher, and the interaction of the interactive label pair causes a change in the fluorescence of the probe. The change in the fluorescence results in a decrease in intensity, a decrease in lifetime, and a change in polarization. 119. The probe of claim 114 , selected from the group consisting of a change in wavelength. The probe according to claim 114 , which is immobilized on a support.   The probe is a bimolecular probe comprising a first molecule including two of the target sequence, the first complementary sequence and the second complementary sequence, and a second molecule including the remaining sequence, The two sequences contained in one molecule are covalently linked by a loop portion, the at least one detectable label includes at least one interactive label pair, and each label pair includes the single sequence The first and second label portions are bonded to each other at different positions of the molecular probe, and when the first or second hybridized duplex is formed, the first and second label portions are mutually connected. The probe according to claim 2 which acts. 119. The probe of claim 118 , wherein at least one of the first and second label portions is covalently linked to the loop portion. The interactive label pair is composed of a phosphor and a quencher, and the interaction of the interactive label pair causes a change in the fluorescence of the probe, and the change in fluorescence is caused by a decrease in intensity, a decrease in lifetime, polarization 119. The probe of claim 118 , selected from the group consisting of a change, a change in wavelength. 119. The probe of claim 118 , wherein the first or second molecule is immobilized on a support. The probe includes a 5 ′ arm sequence covalently linked to the 5 ′ end of the first complementary sequence and a 3 ′ covalently linked to the 3 ′ end of the first complementary sequence. A probe further comprising a nucleic acid arm sequence pair consisting of an arm sequence (3'arm sequence),
The arm sequence pair forms a stem duplex having about 3 to 35 complementary base pairs when the first complementary sequence is not hybridized to the first subject sequence;
The detectable label comprises an interactive label pair consisting of a first label portion joined to the 5 ′ arm sequence and a second label portion joined to the 3 ′ arm sequence, the stem duplex The probe according to claim 2, wherein the first and second label portions interact when a chain is formed. The probe includes a 5 ′ arm sequence covalently linked to the 5 ′ end of the second complementary sequence and a 3 ′ covalently linked to the 3 ′ end of the second complementary sequence. A probe further comprising a nucleic acid arm sequence pair consisting of an arm sequence (3'arm sequence),
The arm sequence pair forms a stem duplex having about 3 to 35 complementary base pairs when the second complementary sequence is not hybridized to the second subject sequence;
The detectable label comprises an interactive label pair consisting of a first label portion joined to the 5 ′ arm sequence and a second label portion joined to the 3 ′ arm sequence, the stem duplex The probe according to claim 2, wherein the first and second label portions interact when a chain is formed. The probe is covalently linked to the first 5 ′ arm sequence covalently linked to the 5 ′ end of the first complementary sequence and the 3 ′ end of the first complementary sequence. A first arm sequence pair consisting of a first 3 ′ arm sequence and a second 5 ′ arm sequence (second) covalently linked to the 5 ′ end of the second complementary sequence. 5'arm sequence) and a second arm sequence pair consisting of a second 3'arm sequence covalently linked to the 3 'end of the second complementary sequence A probe further comprising a nucleic acid arm sequence pair of
When the first complementary sequence is not hybridized to the first target sequence, the first arm sequence pair has a first stem duplex having about 3 to 35 complementary base pairs (first stem duplex). the second arm sequence pair has about 3 to 35 complementary base pairs when the second complementary sequence is not hybridized to the second target sequence. Forming a second stem duplex;
The detectable label comprises a first label pair comprising a first label portion joined to the first 5 ′ arm array and a second label portion joined to the first 3 ′ arm array; Two pairs of interactive labels comprising a third label portion joined to a second 5 'arm arrangement and a second label pair comprising a fourth label portion joined to the second 3' arm arrangement A pair comprising: the first label pair interacts when the first stem duplex is formed and the second label pair interacts when the second stem duplex is formed Item 3. The probe according to Item 2.   The interactive label pair is composed of a phosphor and a quencher, and the interaction of the interactive label pair causes a change in the fluorescence of the probe, and the change in fluorescence results in a decrease in intensity, a decrease in lifetime, and polarization. The probe according to any one of claims 122 to 124, which is selected from the group consisting of a change and a change in wavelength.   The probe according to any one of claims 122 to 124, wherein the probe is a trimolecular probe including a first molecule including the target sequence, a second molecule including the first complementary sequence, and a third molecule including the second complementary sequence. The probe according to one item. 127. A probe according to claim 126 , wherein one of the first, second and third molecules is immobilized on a support.   The probe is a bimolecular probe comprising a first molecule including two of the target sequence, the first complementary sequence and the second complementary sequence, and a second molecule including the remaining sequence, The probe according to any one of claims 157 to 159, wherein the two sequences contained in one molecule are covalently linked by a loop portion. 129. The probe of claim 128 , wherein one of the first and second molecules is immobilized on a support.   The probe is a unimolecular probe, and the first molecule including the target sequence, the second molecule including the first complementary sequence, and the third molecule including the second complementary sequence are shared by at least one loop portion. The probe according to any one of claims 122 to 124, which is connected by a bond. The probe according to claim 130 , which is immobilized on a support. In an assay for detecting the presence or absence of at least one target receptor factor in a sample that can optionally bind to a probe ligand under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 5; and b) checking if there is a change in the magnitude of the characteristic signal compared to when there is no target receptor factor at the detection temperature. Including assay. In an assay for detecting the presence or absence of at least one target ligand in a sample under conditions including a detection temperature,
a) contacting the sample with the probe according to claim 5 in the presence of a receptor factor capable of binding the probe ligand and the target ligand; and b) as compared to when there is no target ligand at the detection temperature. Determining whether there is a change in the magnitude of the characteristic signal. In an assay for detecting the presence or absence of at least one target reaction inducer in a sample capable of specifically cleaving a cleavage site under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 10; and b) checking if there is a change in the magnitude of the characteristic signal compared to when there is no target reaction inducer at the detection temperature. Including assay. In an assay for detecting the presence or absence of at least one target reaction inducer in a sample capable of specifically inducing a covalent bond at a reaction site under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 23 ; and b) checking if there is a change in the magnitude of the characteristic signal compared to when there is no target reaction inducer at the detection temperature. Including assay. In an assay for detecting the presence or absence of at least one target reaction inducer in a sample capable of specifically converting a reaction site to a junction site under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 30 ; and b) checking if there is a change in the magnitude of the characteristic signal compared to when there is no target reaction inducer at the detection temperature. Including assay. In an assay for detecting the presence or absence of at least one target reaction inducer in a sample capable of specifically converting a reactive site to a non-conjugated site under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 43 ; and b) checking if there is a change in the magnitude of the characteristic signal compared to when there is no target reaction trigger at the detection temperature. Including assay. In an assay for detecting an inhibitor or enhancer for binding of a receptor factor to a probe ligand under conditions including a detection temperature,
a) contacting the target compound with the probe according to claim 5 in the presence of the receptor factor; and b) a change in the magnitude of the characteristic signal compared to when there is no target compound at the detection temperature. An assay comprising: In an assay for detecting an inhibitor or enhancer for a reaction inducer capable of specifically cleaving a cleavage site under conditions including a detection temperature,
a) contacting the target compound with the probe according to claim 10 in the presence of the reaction inducer; and b) a change in the magnitude of the characteristic signal compared to when there is no target compound at the detection temperature. An assay comprising the steps of: In an assay for detecting an inhibitor or enhancer against at least one reaction inducer capable of specifically inducing a reactive site covalent bond under conditions including a detection temperature,
a) contacting the target compound with a probe according to claim 23 in the presence of the reaction-inducing factor and destabilizing factor; and b) the characteristic signal compared to when the target compound is absent at the detection temperature. An assay comprising: checking for a change in size. In an assay for detecting an inhibitor or enhancer for at least one reaction inducer capable of specifically converting a reaction site to a junction site under conditions including a detection temperature,
a) contacting the target compound with a probe according to claim 30 in the presence of the reaction inducing factor and destabilizing factor; and b) the characteristic signal compared to when the target compound is absent at the detection temperature. An assay comprising: checking for a change in size. In an assay for detecting an inhibitor or enhancer for at least one reaction inducer capable of specifically converting a reactive site to a non-conjugated site under conditions including a detection temperature,
a) contacting the target compound with the probe according to claim 43 in the presence of the reaction-inducing factor and destabilizing factor; and b) the characteristic signal compared to when the target compound is absent at the detection temperature. An assay comprising: checking for a change in size.   143. The assay according to any one of claims 132 to 142, wherein the step of confirming comprises measuring the magnitude of the characteristic signal. 145. The assay of claim 143 , wherein the step of confirming includes measuring the magnitude of the characteristic signal as a function of time. 138. The assay of any one of claims 132, 133, and 138 , wherein the detection temperature is at least 5 ° C lower than the melting temperature of the first hybridized duplex when the receptor agent is not present. 140. The assay according to any one of claims 134 and 139 , wherein the detection temperature is at least 5 ° C. higher than the melting temperature of the first hybridized duplex in the absence of the reaction inducing agent. 142. The assay according to any one of claims 135, 136, 140 and 141 , wherein the detection temperature is at least 5 ° C lower than the melting temperature of the first hybridized duplex in the absence of the reaction inducer. . 143. The assay according to any one of claims 137 and 142 , wherein the detection temperature is at least 5 ° C higher than the melting temperature of the first hybridized duplex when the reaction inducing agent is not present. 143. The assay according to any one of claims 132 to 142 , wherein the probe is a bimolecular probe or a trimolecular probe having at least one non-interactive label. 149. The assay of claim 149 , wherein the probe is not an immobilized probe and the step of identifying comprises separating a species that has not hybridized with a hybridized species. 149. The assay of claim 149 , wherein the probe is an immobilized probe and the step of confirming comprises washing unbound species. 143. The assay according to any one of claims 132 to 142 , wherein the detectable label is an intercalating dye. 157. The assay of claim 152 , wherein the probe is not an immobilized probe and the step of identifying comprises separating a species that has not hybridized with a hybridized species. 157. The assay of claim 152 , wherein the probe is an immobilized probe and the step of confirming comprises washing unbound species. 143. The assay according to any one of claims 132 to 142 , wherein the probe is a probe comprising at least one interactive label pair. 165. The assay of claim 155 , wherein the interactive label pair is a fluorophore and quencher pair or a phosphor and quencher pair. The assay of claim 155 , wherein said probe is a unimolecular probe. The assay of claim 155 , wherein said probe is a bimolecular probe. The assay of claim 155 , wherein said probe is a trimolecular probe. 165. The assay of claim 155 , wherein the probe is an immobilized probe. 138. The assay according to any one of claims 132 to 137 , wherein the probe is a unimolecular probe comprising at least one interactive label pair consisting of a phosphor and a quencher, or a light emitter and a quencher. 163. The assay of claim 161 , wherein the sample is a cell or tissue and the contacting comprises introducing the probe into or outside the cell or tissue. 139. The assay of claim 138 , wherein the probe is a unimolecular probe comprising at least one interactive label pair consisting of a fluorophore and a quencher, or a phosphor and a quencher. 166. The assay of claim 163 , wherein the sample is a cell or tissue and the contacting comprises introducing the probe and the target compound into or out of the cell or tissue. 165. The assay of claim 164 , wherein the receptor factor is a receptor present inside or outside the cell or tissue. 165. The assay of claim 164 , wherein the receptor factor is the receptor protein expressed recombinantly within the cell or tissue by introducing a vector encoding the receptor protein. 143. The assay according to any one of claims 139 to 142 , wherein the probe is a unimolecular probe comprising at least one interactive label pair consisting of a phosphor and a quencher, or a light emitter and a quencher. 167. The assay of claim 167 , wherein the sample is a cell or tissue and the contacting step comprises introducing the probe and the target compound into or outside the cell or tissue. 169. The assay of claim 168 , wherein said reaction inducing factor is an enzyme present inside or outside of said cell or tissue. 169. The assay of claim 169 , wherein said reaction inducer is an enzyme that is recombinantly expressed within the cell or tissue by introducing a vector encoding the enzyme. In an assay for detecting the presence or absence of at least one target receptor factor in a sample that can optionally bind to a probe ligand under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 54 ; and
b) checking whether there is a change in the magnitude of the characteristic signal compared to when there is no target receptor factor at the detection temperature. In an assay for detecting the presence or absence of at least one target ligand in a sample under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 54 in the presence of a receptor factor capable of binding the probe ligand and the target ligand;
b) confirming whether there is a change in the magnitude of the characteristic signal compared to when there is no target ligand at the detection temperature. In an assay for detecting the presence or absence of at least one target reaction inducer in a sample capable of specifically cleaving a cleavage site under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 56 ; and
b) confirming whether there is a change in the magnitude of the characteristic signal compared to when there is no target reaction inducer at the detection temperature. In an assay for detecting the presence or absence of at least one target reaction inducer in a sample capable of specifically inducing a covalent bond at a reaction site under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 58 ; and
b) confirming whether there is a change in the magnitude of the characteristic signal compared to when there is no target reaction inducer at the detection temperature. In an assay for detecting the presence or absence of at least one target reaction inducer in a sample capable of specifically converting a reaction site to a junction site under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 60 ; and
b) confirming whether there is a change in the magnitude of the characteristic signal compared to when there is no target reaction inducer at the detection temperature. In an assay for detecting the presence or absence of at least one target reaction inducer in a sample capable of specifically converting a reactive site to a non-conjugated site under conditions including a detection temperature,
a) contacting the sample with a probe according to claim 62 ; and
b) confirming whether there is a change in the magnitude of the characteristic signal compared to when there is no target reaction inducer at the detection temperature. In an assay for detecting an inhibitor or enhancer for binding of a receptor factor to a probe ligand under conditions including a detection temperature,
contacting a target compound with a probe according to claim 54 in the presence of said receptor factor; and
b) confirming whether there is a change in the magnitude of the characteristic signal compared to when there is no target candidate compound at the detection temperature. In an assay for detecting an inhibitor or enhancer for a reaction inducer capable of specifically cleaving a cleavage site under conditions including a detection temperature,
contacting a target compound with a probe according to claim 56 in the presence of said reaction inducer; and
b) checking whether there is a change in the magnitude of the characteristic signal compared to when there is no target compound at the detection temperature. In an assay for detecting an inhibitor or enhancer against at least one reaction inducer capable of specifically inducing a reactive site covalent bond under conditions including a detection temperature,
contacting a target compound with a probe according to claim 58 in the presence of said reaction-inducing factor and destabilizing factor; and
b) checking whether there is a change in the magnitude of the characteristic signal compared to when there is no target compound at the detection temperature. In an assay for detecting an inhibitor or enhancer for at least one reaction inducer capable of specifically converting a reaction site to a junction site under conditions including a detection temperature,
a) contacting the target compound with a probe according to claim 60 in the presence of said reaction inducer and destabilizing factor; and
b) checking whether there is a change in the magnitude of the characteristic signal compared to when there is no target compound at the detection temperature. In an assay for detecting an inhibitor or enhancer for at least one reaction inducer capable of specifically converting a reactive site to a non-conjugated site under conditions including a detection temperature,
a) contacting the target compound with a probe according to claim 62 in the presence of said reaction inducer and destabilizing factor; and
b) checking whether there is a change in the magnitude of the characteristic signal compared to when there is no target compound at the detection temperature.   An inhibitor which detects at least one probe according to any one of claims 1 and 2 and at least one target factor or target ligand or which inhibits or enhances the interaction of the target factor and the recognition element or Instructions for conducting an assay to detect the enhancer. From the group consisting of a salt, a buffer solution, a nuclease inhibitor, a substrate for an enzyme or enzyme-binding label, a receptor factor, a reaction inducer, a vector encoding a receptor protein or enzyme that acts as a target factor, and a ligation reagent 183. The kit of claim 182 , further comprising one or more selected reagents. A kit further comprising one or more components suitable for performing an in vivo or in situ assay, said components comprising the group consisting of a permeabilizing agent and a ribosome precursor 183. The kit according to claim 182 , which is a component selected and necessary for introducing the probe into a cell. 183. The kit according to claim 182 , wherein the probe is an immobilized probe. 186. The kit of claim 185 , comprising at least one additional immobilized probe having different targeting factors and immobilized at a predetermined location on the same support. 186. The kit of claim 185 , comprising at least one additional immobilized probe having the same target agent and immobilized at different predetermined locations on the same support.   A target detection system comprising at least one probe according to claim 1 or 2.   A pre-probe comprising at least one, preferably all components, operably linked:
  a) a first pair of nucleic acid sequences comprising a first subject sequence and a first complementary sequence, wherein the first subject sequence and the first complementary sequence are each independently about 3 to about 150 nucleotides And are substantially complementary to each other to form a first hybridized duplex;
  b) at least one reactive group joined to at least one of the first subject sequence and the first complementary sequence, wherein the reactive group specifically interacts with at least one target element; A possible recognition element is a chemical moiety that can be joined; and
  c) Optionally, at least one detectable label, wherein the detectable label produces a characteristic signal whose level is a function of the amount of the first hybridized duplex.   189. The preprobe of claim 189, further comprising a second pair of competing nucleic acid sequences consisting of a second subject sequence and a second complementary sequence,
  The second subject sequence and the second complementary sequence each independently comprise from about 3 to about 150 nucleotides and are substantially complementary to each other to form a second hybridized duplex; and
  The said 1st and 2nd object sequence is a preprobe which is contained in one object sequence, and has the overlap region which consists of at least 1 nucleotide.   191. The preprobe of claim 190, wherein the reactive group is conjugated to the first subject sequence or the first complementary sequence that does not include the overlap.   191. The preprobe of claim 189 or 190, wherein the reactive group is selected from the group consisting of hydroxyl, carboxyl, amino, sulfhydryl, aldehyde, and phosphate.   The pre-probe according to claim 189 or 190, wherein the recognition element is a probe ligand capable of specifically binding to a receptor factor.   191. The preprobe of claim 189 or 190, wherein the recognition element is a probe substrate comprising at least one cleavage site that can be specifically cleaved by a reaction inducing factor and a destabilizing factor.   191. The preprobe of claim 189 or 190, wherein the recognition element is a probe substrate comprising at least one reaction site to which a destabilizing factor can be specifically conjugated by a reaction inducer.   191. The preprobe of claim 189 or 190, wherein the recognition element is a probe substrate comprising at least one reaction site that can be specifically converted by a reaction inducing factor into a conjugation site to which a destabilizing factor can be joined.   191. The probe substrate according to claim 189 or 190, wherein the recognition element is a probe substrate that includes at least one reaction site that can be converted to a non-bonding site by a reaction inducing factor, which is a bonding site to which a destabilizing factor can be bonded. Pre-probe.   190. A kit comprising at least one preprobe according to claim 189 or 190 and instructions for joining the recognition element to the reaction site.