Methods for detecting intermolecular interactions in vivo and in vitro

Abstract

Methods for assessing intermolecular interactions in vivo and in vitro are provided. Methods are provided for detecting protein-DNA interactions in vivo, in which a cell having a chimeric guide endonuclease molecule and a target nucleic acid is provided, and cleavage of the target nucleic acid by the chimeric guide endonuclease molecule is monitored. Cleavage by the chimeric guide molecule corresponds to binding of the guide domain to the target nucleic acid, or to a protein associated with the nucleic acid. The methods of the invention are adapted to cleavage of target nucleic acids, amplification of target nucleic acids, detection of target nucleic acids, screening of genomic target nucleic acid sequences for guide binding domains, and screening for modulators of chimeric guide binding domain activity. Also provided are methods for detecting interactions between other molecules, including hormones and receptors, enzymes and substrates, and the like.

What is claimed is:  
     
         1 . A method of cleaving a target nucleic acid in a cell with a chimeric guide-endonuclease fusion molecule, the method comprising: 
 (i) providing a cell comprising the target nucleic acid and a chimeric guide molecule-endonuclease fusion molecule; and,    (ii) permitting the fusion molecule to cleave the target nucleic acid, thereby producing a cleaved target nucleic acid with a site of cleavage.    
     
     
         2 . The cell of  claim 1 , wherein the cell comprises a nucleic acid which encodes the chimeric guide molecule-endonuclease fusion molecule, which fusion molecule is a protein, and the method further comprises the step of expressing the chimeric guide molecule-endonuclease fusion molecule in the cell, thereby providing the cell comprising the target nucleic acid and a chimeric guide molecule-endonuclease fusion molecule.  
     
     
         3 . The method of  claim 2 , further comprising transducing the cell with the chimeric nucleic acid encoding the chimeric guide molecule-endonuclease fusion molecule.  
     
     
         4 . The method of  claim 2 , further comprising co-transducing the cell with the chimeric nucleic acid encoding the chimeric guide molecule-endonuclease fusion molecule and the target nucleic acid, and optionally comprising the step of transducing the cell with a marker nucleic acid.  
     
     
         5 . The method of  claim 2 , further comprising the steps of transducing the cell with a nucleic acid encoding a selectable component, and selecting transduced cells using the selectable component, wherein expression of the selectable component corresponds to transduction of the cell by the chimeric nucleic acid encoding the guide molecule-endonuclease chimeric molecule.  
     
     
         6 . The method of  claim 1 , further comprising detecting the cleaved nucleic acid.  
     
     
         7 . The method of  claim 1 , further comprising amplifying the cleaved target nucleic acid.  
     
     
         8 . The method of  claim 1 , further comprising the steps of amplifying the cleaved target nucleic acid and detecting the amplified target nucleic acid.  
     
     
         9 . The method of  claim 1 , further comprising transducing the cell with the target nucleic acid.  
     
     
         10 . The method of  claim 1 , further comprising the step of detecting the cleaved nucleic acid, wherein the target nucleic acid is detected using a technique selected from the group consisting of nested PCR, Southern blotting, northern blotting, and, cloning and sequencing the target nucleic acid.  
     
     
         11 . The method of  claim 1 , wherein the cell is embedded in agarose to reduce shearing of the target nucleic acid.  
     
     
         12 . The method of  claim 1 , wherein the target nucleic acid is selected from the group consisting of a genomic nucleic acid with a known sequence of nucleotides, a genomic nucleic acid with an unknown sequence of nucleotides, a plasmid with a sequence of known nucleotides, a plasmid with a sequence of unknown nucleotides, an RNA with a sequence of known nucleotides, and an RNA with an unknown sequence of nucleotides.  
     
     
         13 . The method of  claim 1 , wherein the chimeric guide-molecule endonuclease fusion molecule is a calcium inducible protein, and the method further comprises the step of adding exogenous calcium to the cell, thereby inducing the protein to cleave the target nucleic acid in step (ii).  
     
     
         14 . The method of  claim 1 , wherein the endonuclease is derived from micrococcal nuclease.  
     
     
         15 . The method of  claim 1 , wherein the guide domain is derived from the group of proteins consisting of a DNA binding protein, an RNA binding protein, a protein which binds to a DNA binding protein, a protein which binds to an RNA binding protein, an antibody protein which binds to a DNA binding protein, a first antibody protein which binds to a second antibody protein, an antibody protein which binds to an RNA binding protein, and, a guide nucleic acid which hybridizes to the target nucleic acid.  
     
     
         16 . The method of  claim 1 , wherein cleavage of the target nucleic acid by the guide endonuclease chimera produces a 3′ phosphate and a 5′ hydroxyl at the site of cleavage.  
     
     
         17 . The method of  claim 1 , further comprising the step of ligating an oligonucleotide to the cleaved target nucleic acid.  
     
     
         18 . The method of  claim 1 , further comprising the steps of: 
 (a) making a first double-stranded target DNA and, optionally, a second double-stranded target DNA which correspond to each side of the cleavage site;    (b) ligating a first oligonucleotide comprising a first class IIS restriction site to the first double-stranded target DNA, thereby producing a first ligated target DNA, the first oligonucleotide optionally further comprising a first class I restriction site and a first detectable label;    (c) optionally, ligating a second oligonucleotide comprising a second class IIS restriction site to the second double-stranded target DNA, thereby producing a second ligated target DNA, the second oligonucleotide optionally further comprising a first class I restriction site and a second detectable label;    (d) cleaving the first ligated target DNA and, optionally, cleaving the second ligated target DNA with the first and, optionally, the second class IIS restriction enzymes; and    (e) isolating the first and, optionally, the second ligated target DNAs, optionally by capture of the first and second detectable label and cleavage of the captured label with the first and second class I restriction enzyme.    
     
     
         19 . The method of  claim 18 , further comprising the steps of: 
 (f) ligating the first and second ligated target DNAs to form a ligated target site; and optionally comprising the steps of:    (g) concatemerizing the ligated target site to produce a concatemerized target site;    (h) cloning the concatemerized the target site into a nucleic acid vector to produce a cloned target site; and,    (i) sequencing the cloned target site.    
     
     
         20 . The method of  claim 19 , further comprising the steps of: 
 (j) 3′ dephosphorylation of the 3′ phosphate at the site of cleavage, thereby producing a 3′ dephosphorylated cleavage end;    (k) extending the dephosphorylated cleavage end, thereby producing an extended 3′ end; and,    (l) PCR amplifying the extended 3′ end.    
     
     
         21 . The method of  claim 1 , wherein the target nucleic acid is a plasmid nucleic acid and the chimeric guide domain is a calcium inducible polypeptide domain, the method further comprising the steps of: 
 (a) permeabilizing the cell and treating the cell with calcium, thereby inducing the calcium inducible chimeric molecule, which molecule cleaves at least one strand of the plasmid, leaving a 3′ phosphate and a 5′ hydroxyl the site of cleavage;    (b) isolating plasmid nucleic acids from the cell, including the cleaved plasmid, thereby producing isolated plasmid nucleic acids,    (c) primer extending the cleaved plasmid using a primer extension primer which is complementary to a strand of the plasmid comprising the cleavage site, thereby producing a double-stranded blunt end at the site of cleavage;    (d) ligating a trapping oligonucleotide to the double-stranded blunt end;    (e) PCR amplifying the cleaved plasmid using a PCR reaction mixture comprising an oligonucleotide which hybridizes to the trapping oligonucleotide, and, optionally, the primer extension primer, thereby amplifying the nucleic acid; and, optionally, further comprising the step of: 
 (f) cloning the guide molecule-chimera, thereby producing a chimeric nucleic acid, wherein an endonuclease cleavage domain of the chimeric molecule is calcium inducible, cleaves a single strand of the plasmid, and produces a 3′ phosphate and a 5′ hydroxyl at the site of cleavage.  
   
     
     
         22 . The method of  claim 21 , further comprising the steps of: 
 (g) 3′ dephosphorylating a 3′ phosphate at the site of cleavage, thereby producing a 3′ dephosphorylated cleavage end;    (j) extending the dephosphorylated cleavage end, thereby producing an extended 3′ end; and,    (k) PCR amplifying the extended 3′ end.    
     
     
         23 . The method of  claim 21 , wherein an internal PCR primer is used in step (e.) to prime the PCR.  
     
     
         24 . The method of  claim 21 , further comprising detecting the PCR amplified nucleic acid.  
     
     
         25 . The method of  claim 1 , wherein cleavage of the target nucleic acid by the guide molecule-endonuclease chimera produces a 3′ phosphate and a 5′ hydroxyl at the site of cleavage, further comprising one or more step selected from the steps of: 
 (a) 5′ phosphorylating the cleaved nucleic acid at the site of cleavage to produce a 5′ phosphorylated site;  
 (b) 3′ dephosphorylating the cleaved nucleic acid at the site of cleavage;  
 (c) extending the 3′ end of the cleavage site with a terminal transferase enzyme;  
 (d) extending the 3′ end of the cleavage site by ligating a 3′ extension oligonucleotide to the cleaved nucleic acid;  
 (e) primer extending the cleaved target nucleic acid using a primer extension primer which is complementary to a strand of the nucleic acid comprising the cleavage site, thereby producing a double-stranded blunt end at the site of cleavage;  
 (f) extending the 5′ end by ligating a 5′ extension oligonucleotide to the cleaved target DNA;  
 (g) PCR amplifying target nucleic acid using a primer complementary to the 5′ extension oligonucleotide; and,  
 (h) performing nested PCR on amplified nucleic acids.  
 
     
     
         26 . The method of  claim 1 , wherein cleavage of the target nucleic acid by the guide molecule-endonuclease chimera produces a 3′ phosphate and a 5′ hydroxyl at the site of cleavage, further comprising one or more step selected from the steps of: 
 (a) 5′ phosphorylating the cleaved nucleic acid at the site of cleavage to produce a 5′ phosphorylated site;  
 (b) 3′ dephosphorylating the cleaved nucleic acid at the site of cleavage;  
 (c) extending the 3′ end of the cleavage site with a terminal transferase enzyme;  
 (d) extending the 3′ end of the cleavage site by ligating a 3′ extension oligonucleotide to the cleaved nucleic acid;  
 (e) primer extending the cleaved target nucleic acid using a primer extension primer which is complementary to a strand of the nucleic acid comprising the cleavage site, thereby producing a double-stranded blunt end at the site of cleavage;  
 (f) extending the 5′ end by ligating a 5′ extension oligonucleotide to the cleaved target DNA;  
 (g) PCR amplifying target nucleic acid using a primer complementary to the 5′ extension oligonucleotide; and,  
 (h) performing nested PCR on amplified nucleic acids.  
 
     
     
         27 . The method of  claim 1 , wherein the target nucleic acid is a genomic DNA, the chimeric molecule is calcium inducible, and the method further comprises the steps of: 
 (a) co-transducing the cell with a marker vector and a chimeric nucleic acid vector encoding the chimeric guide molecule, and culturing the cell under conditions which permit expression of a marker encoded by the marker vector and the chimeric guide molecule, thereby producing a marked cell which expresses the marker and the chimeric guide molecule;    (b) isolating the marked cell, thereby providing an isolated cell;    (c) permeabilizing the isolated cell with a mild detergent and treating the isolated cell with calcium, thereby inducing the calcium inducible chimeric molecule, which molecule cleaves at least one strand of the target nucleic acid, leaving a 3′ phosphate and a 5′ hydroxyl the site of cleavage; and,    (d) further performing one or more step selected from the steps of:    (e) 5′ phosphorylating the 5′ hydroxyl of step (c), thereby providing a 5′ phosphate site on the cleaved nucleic acid for primer extension;    (f) ligating a trapping oligonucleotide to the 5′ phosphate site to produce a target-linker nucleic acid;    (g) 3′ dephosphorylating the 3′ phosphate at the site of cleavage;    (h) 3′ end extending the 3′ cleavage site with a terminal transferase enzyme to produce a first extended-target nucleic acid;    (i) 3′ end extending the 3′ cleavage site with by ligation of an oligonucleotide to produce a second extended-target nucleic acid;    (j) PCR amplifying the target-linker nucleic acid of step (f), to produce an amplified target-linker nucleic acid;    (k) 3′ end extension of the amplified target-linker nucleic acid;    (l) PCR amplification of the first extended-target nucleic acid of step (h) to produce a first amplified extended-target nucleic acid;    (m) PCR amplification of the second extended target nucleic acid of step (i) to produce a second amplified extended-target nucleic acid; and,    (n) detection of a nucleic acid selected from the group consisting of the first amplified extended-target nucleic acid of step (l), the second amplified extended-target nucleic acid of step (m) and the amplified target-linker nucleic acid of step (j).    
     
     
         28 . The method of  claim 1 , wherein the target nucleic acid is an RNA, wherein cleavage of the RNA by the guide molecule-endonuclease chimera produces a 3′ phosphate and a 5′ hydroxyl at the site of cleavage and the method further comprises the steps of: 
 (a) isolating RNA from the cell, thereby producing isolated RNA;  
 (b) coupling an RNA terminator to 3′ hydroxyls present in the isolated RNA; and,  
 (c) dephosphorylating the 3′ phosphate to produce a dephosphorylated target RNA, using an enzyme selected from the group consisting of a kinase enzyme, and a phosphatase enzyme.  
 
     
     
         29 . The method of  claim 28 , further comprising one or more steps selected from the steps consisting of: 
 (d) ligating a first oligonucleotide to the dephosphorylated target RNA, which first oligonucleotide optionally comprises a binding site for a class IIS restriction enzyme;    (e) ligating a second oligonucleotide to the dephosphorylated target RNA which second oligonucleotide optionally comprises a binding site for a class IIS restriction enzyme;    (f) reverse transcribing the cleaved target nucleic acid, optionally using a reverse transcription primer which hybridizes to the first oligonucleotide of step (d) to prime the reverse transcription reaction, thereby producing a reverse transcribed nucleic acid;    (g) treating the reverse transcribed nucleic acid of step (e) with a first enzyme with RNAse H activity, and a second enzyme with DNA polymerase activity to make a double-stranded target DNA;    (h) cleaving the double-stranded DNA with a restriction enzyme, thereby producing a restricted target DNA;    (i) ligating a third oligonucleotide to the restricted target DNA;    (j) isolating the restricted target DNA, thereby producing an isolated restricted target DNA;    (k) PCR amplifying the isolated restricted target DNA, thereby producing amplified restricted target DNA; and,    (l) cloning the restricted amplified target DNA.    
     
     
         30 . The method of  claim 28 , further comprising one or more steps selected from the steps consisting of: 
 (d) ligating a first oligonucleotide to the dephosphorylated target RNA, which first oligonucleotide optionally comprises a binding site for a class IIS restriction enzyme;    (e) ligating a second oligonucleotide to the dephosphorylated target RNA which second oligonucleotide optionally comprises a binding site for a class IIS restriction enzyme;    (f) reverse transcribing the cleaved target nucleic acid, optionally using a reverse transcription primer which hybridizes to the first oligonucleotide of step (d) to prime the reverse transcription reaction, thereby producing a reverse transcribed nucleic acid;    (g) treating the reverse transcribed nucleic acid of step (e) with a first enzyme with RNAse H activity, and a second enzyme with DNA polymerase activity to make a double-stranded target DNA;    (h) cleaving the double-stranded DNA with a restriction enzyme, thereby producing a restricted target DNA;    (i) ligating a third oligonucleotide to the restricted target DNA;    (j) isolating the restricted target DNA, thereby producing an isolated restricted target DNA;    (k) PCR amplifying the isolated restricted target DNA, thereby producing amplified restricted target DNA; and,    (l) cloning the restricted amplified target DNA.    
     
     
         31 . The method of  claim 1 , wherein the method further comprises one or more step selected from the steps of: 
 (a) random primer extending the cleaved target nucleic acid;    (b) ligating a blocking oligonucleotide to the cleaved target nucleic acid;    (c) 5′ phosphorylating the cleaved target nucleic acid;    (d) cleaving the target nucleic acid with a restriction enzyme;    (e) cleaving an amplified target nucleic acid with a restriction enzyme;    (f) trapping cleaved target DNA to produce trapped target DNA;    (g) ligating a reaching oligonucleotide to the trapped target DNA;    (h) amplifying the target DNA using PCR;    (i) blocking a 3′OH terminal of the cleaved target nucleic acid with a terminal transferase;    (j) blocking a 3′OH terminal of the cleaved target nucleic acid with a blocking oligonucleotide;    (k) 3′ dephosphorylating the cleaved target nucleic acid;    (l) 3′ end extending the cleaved target nucleic acid with a terminal transferase enzyme;    (m) 3′ end extending the cleaved target nucleic acid by ligating an oligonucleotide onto the 3′ end of a cleaved target nucleic acid;    (n) primer extending the cleaved target nucleic acid;    (o) cloning the guide molecule-chimera; and,    (p) combining one or more of the preceding steps (a)-(o).    
     
     
         32 . The method of  claim 1 , wherein the method further comprises one or more step selected from the steps of: 
 (a) random primer extending the cleaved target nucleic acid;    (b) ligating a blocking oligonucleotide to the cleaved target nucleic acid;    (c) 5′ phosphorylating the cleaved target nucleic acid;    (d) cleaving the target nucleic acid with a restriction enzyme;    (e) cleaving an amplified target nucleic acid with a restriction enzyme;    (f) trapping cleaved target DNA to produce trapped target DNA;    (g) ligating a reaching oligonucleotide to the trapped target DNA;    (h) amplifying the target DNA using PCR;    (i) blocking a 3′OH terminal of the cleaved target nucleic acid with a terminal transferase;    (j) blocking a 3′OH terminal of the cleaved target nucleic acid with a blocking oligonucleotide;    (k) 3′ dephosphorylating the cleaved target nucleic acid;    (l) 3′ end extending the cleaved target nucleic acid with a terminal transferase enzyme;    (m) 3′ end extending the cleaved target nucleic acid by ligating an oligonucleotide onto the 3′ end of a cleaved target nucleic acid;    (n) primer extending the cleaved target nucleic acid;    (o) cloning the guide molecule-chimera; and,    (p) combining one or more of the preceding steps (a)-(o).    
     
     
         33 . The method of  claim 1 , wherein the method further comprises fixing and permeabilizing the cell.  
     
     
         34 . A method of screening test nucleic acids for in vivo cleavage sites which are cleaved by a chimeric guide molecule, comprising: 
 (A) providing a cell comprising a chimeric nucleic acid encoding the chimeric guide molecule and a test nucleic acid;    (B) permitting the chimeric nucleic acid to be expressed in the cell, thereby producing chimeric guide molecule in the cell;    (C) incubating the cell under conditions in which the guide molecule is active, and,    (D) determining whether the chimeric guide molecule cleaves the test nucleic acid, thereby determining whether the test nucleic acid comprises an in vivo cleavage site for the chimeric guide molecule.    
     
     
         35 . The method of  claim 34 , wherein the test nucleic acid encodes a promoter sequence operably linked to a reporter gene, and the method further comprises detection of the presence or absence of reporter gene expression, which expression is an indicator for whether the test nucleic acid comprises an in vivo cleavage site for the chimeric guide molecule.  
     
     
         36 . The method of  claim 34 , further comprising the step of co-transducing a cell with a first plasmid encoding the target nucleic acid and a second plasmid encoding the chimeric guide molecule.  
     
     
         37 . The method of  claim 34 , further comprising transducing the cell with a second test nucleic acid and determining whether the chimeric guide molecule cleaves the second test nucleic acid, thereby determining whether the second test nucleic acid comprises an in vivo cleavage site for the chimeric guide molecule.  
     
     
         38 . The method of  claim 34 , further comprising, in parallel with steps (A)-(D), performing the steps of: 
 (E) providing a second cell comprising a second chimeric nucleic acid encoding a second chimeric guide molecule and a second test nucleic acid;    (F) permitting the second chimeric nucleic acid to be expressed in the second cell, thereby producing the second chimeric guide molecule in the second cell;    (G) incubating the second cell under conditions in which the second guide molecule is active, and,    (H) determining whether the second chimeric guide molecule cleaves the second test nucleic acid, thereby determining whether the second test nucleic acid comprises an in vivo cleavage site for the chimeric guide molecule.    
     
     
         39 . The method of  claim 31 , wherein the cell further comprises a control nucleic acid which is cleaved by the chimeric guide molecule, and the method further comprises the steps of: 
 (E) permitting the control nucleic acid to be cleaved in the cell during step (C); and,    (F) comparing the rate of cleavage of the test nucleic acid and the control nucleic acid, thereby determining the affinity of the guide molecule for the test nucleic acid, as compared to the control nucleic acid.    
     
     
         40 . A method of detecting a chimeric guide endonuclease molecule modulating agent, comprising the steps of: 
 providing a cell comprising a test nucleic acid, which cell expresses a chimeric guide-endonuclease molecule;    contacting the cell with the modulating agent; and,    measuring the rate of cleavage of the test nucleic acid by the chimeric guide endonuclease molecule in the presence of the agent.    
     
     
         41 . The method of  claim 40 , wherein the target nucleic acid is a promoter sequence which is bound by a transcription factor.  
     
     
         42 . The method of  claim 40 , further comprising the step of comparing the rate of cleavage of the test nucleic acid in the presence of the agent to the rate of cleavage of the test nucleic in an absence of the agent.  
     
     
         43 . The method of  claim 40 , wherein the cell further comprises a second test nucleic acid binding site and the method further comprises the steps of measuring the rate of cleavage of the second test nucleic acid in the presence of the agent.  
     
     
         44 . The method of  claim 40 , further comprising the steps of: 
 providing a second cell comprising a second test nucleic acid;    contacting the second cell with the modulating agent; and,    measuring the rate of cleavage of the second test nucleic acid by the chimeric molecule in the presence of the agent.    
     
     
         45 . The method of  claim 44 , wherein the second cell is contacted with the modulating agent in parallel with contacting the first cell with the modulating agent.  
     
     
         46 . A method of detecting whether a first molecule is in close proximity to a second molecule, the method comprising the steps of: 
 attaching a molecular beacon to the first molecule, wherein the molecular beacon comprises an oligonucleotide to which is attached a fluorophore and a quencher;    attaching an endonuclease moiety to the second molecule; and    determining whether the first molecule is in close proximity to the second molecule by detecting whether fluorescence is emitted by the fluorophore, wherein fluorescence emission is indicative of cleavage of the oligonucleotide by the endonuclease moiety, thereby causing separation of the fluorophore and the quencher.    
     
     
         47 . The method of  claim 46 , wherein either or both of the first molecule and the second molecule are located in a cell.  
     
     
         48 . The method of  claim 46 , wherein either or both of the first molecule and the second molecule are located in an organism.  
     
     
         49 . The method of  claim 46 , wherein the target molecule is present in a tissue sample.  
     
     
         50 . The method of  claim 46 , wherein the molecular beacon is covalently attached to the first molecule.  
     
     
         51 . The method of  claim 46 , wherein the molecular beacon is noncovalently attached to the first molecule.  
     
     
         52 . The method of  claim 51 , wherein the molecular beacon is attached to a binding moiety which binds to the first molecule.  
     
     
         53 . The method of  claim 52 , wherein the binding moiety is an antibody.  
     
     
         54 . The method of  claim 46 , wherein the endonuclease moiety is covalently attached to the second molecule.  
     
     
         55 . The method of  claim 46 , wherein the endonuclease moiety is noncovalently attached to the second molecule.  
     
     
         56 . The method of  claim 55 , wherein the endonuclease moiety comprises a binding moiety which binds to the second molecule.  
     
     
         57 . The method of  claim 56 , wherein the binding moiety is an antibody.  
     
     
         58 . The method of  claim 46 , wherein the first molecule and the second molecule are separated by about ten nanometers or less.  
     
     
         59 . The method of  claim 58 , wherein the first molecule is in contact with the second molecule.  
     
     
         60 . The method of  claim 46 , wherein the endonuclease moiety comprises a calcium-inducible endonuclease, and the method further comprises the step of contacting the endonuclease moiety with calcium.  
     
     
         61 . The method of  claim 46 , wherein the first molecule comprises a first member of a binding pair and the second molecule comprises a second member of the binding pair, and wherein the binding pair is selected from the group consisting of: enzyme:substrate, hormone:ligand, drug:receptor, protein:protein, protein/modifier, protein:nucleic acid; and nucleic acid:nucleic acid.  
     
     
         62 . The method of  claim 46 , wherein the emission of fluorescence is detected by fluorescent microscopy or fluorometry.  
     
     
         63 . A method of detecting a target molecule, the method comprising the steps of: 
 contacting the target molecule with a chimeric endonuclease which binds to the target molecule;    contacting the chimeric endonuclease with a molecular beacon comprising an oligonucleotide to which is attached a fluorophore and a quencher; and    detecting the presence of a fluorescent signal which results from cleavage of the oligonucleotide by the endonuclease, thereby allowing separation of the quencher from the fluorophore.    
     
     
         64 . The method of  claim 63 , wherein the target molecule is present in a cell.  
     
     
         65 . The method of  claim 63 , wherein the target molecule is present in a tissue sample.  
     
     
         66 . The method of  claim 63 , wherein the fluorescent signal is integrated over time.  
     
     
         67 . The method of  claim 63 , wherein the chimeric endonuclease comprises a calcium-inducible endonuclease moiety, and the method further comprises the step of contacting the endonuclease moiety with calcium.  
     
     
         68 . The method of  claim 63 , wherein the chimeric endonuclease binds directly to the target molecule.  
     
     
         69 . The method of  claim 68 , wherein the target molecule is a nucleic acid and the chimeric endonuclease comprises a nucleic acid binding domain.  
     
     
         70 . The method of  claim 63 , wherein the chimeric endonuclease binds indirectly to the target molecule.  
     
     
         71 . The method of  claim 70 , wherein a primary binding moiety binds to the target molecule and the chimeric endonuclease binds to the primary binding moiety.  
     
     
         72 . The method of  claim 71 , wherein the primary binding moiety is an antibody that binds to the target molecule and the chimeric endonuclease comprises a moiety that binds to the antibody.