Probe for detection and quantification of nitric oxide, and method for detecting and quantifying nitric oxide using the same

Abstract

Probe 1 for detection and quantification of nitric oxide, which, comprises two subunits 21 and 22 of soluble guanylate cyclase 2 and cGMP-visualization probes 3 respectively connected with each subunit, wherein the cGMP-visualization probe generates signal upon recognizing guanosine 3′ , 5 -cyclic monophospate.

1 . A probe for detection and quantification of nitric oxide, which comprises two subunits of soluble guanylate cyclase and cGMP-visualization probes respectively connected with each subunit, wherein the cGMP-visualization probe generates signal upon recognizing guanosine 3′,5′-cyclic monophospate. 
     
     
         2 . The probe of  claim 1 , which is a dimmer of hybrid proteins, wherein the hybrid proteins are α- and β-subunits of soluble guanylate cyclase respectively connecting with the cGMP-visualization probe. 
     
     
         3 . The probe of  claim 1 , wherein the cGMP-visualization probe comprises cGMP-binding protein and two reporters connected with each end of the cGMP-binding protein so that the approach of the two reporters becomes detectable. 
     
     
         4 . The probe of  claim 3 , wherein the cGMP-binding protein is cGMP-dependent protein kinase Iα. 
     
     
         5 . The prove of  claim 3 , wherein the two reporters are yellow fluorescent protein and cyan fluorescent protein. 
     
     
         6 . A method for detecting and quantifying nitric oxide, which comprises making the probe of  claim 1  to coexist with guanosine 5′-triphospate, and measuring signal change. 
     
     
         7 . The method according to  claim 6 , wherein the probe coexists with guanosine 5′-triphospate in a cell by introducing a polynucleotide expressing the probe into the cell. 
     
     
         8 . The method according to  claim 6 , wherein the probe coexists with guanosine 5′-triphospate in a cell by introducing a pair of polynucleotides respectively expressing a hybrid protein into the cell, wherein each of the hybrid protein comprises α- or β-subunits of soluble guanylate cyclase connecting with the cGMP-visualization probe. 
     
     
         9 . The method according to  claim 6 , wherein the probe coexists with guanosine 5′-triphospate in all cells of a non-human animal or its progeny established by introducing polynucleotide expressing the probe into a non-human totipotent cell and developing the cell to individual. 
     
     
         10 . The method according to  claim 6 , wherein the probe coexists with guanosine 5′-triphospate in all cells of a non-human animal or its progeny established by introducing a pair of polynucleotides respectively expressing a hybrid protein into the cell, wherein each of the hybrid protein comprises α- or β-subunits of soluble guanylate cyclase connecting with the cGMP-visualization probe. 
     
     
         11 . A method for screening a substance acting on binding of nitric oxide to soluble guanylate cyclase, which comprises making the probe of  claim 1  coexist with guanosine 5′-triphospate, a candidate substance and nitric oxide, and measuring signal change with and without the candidate substance. 
     
     
         12 . The method according to  claim 11 , wherein the probe coexists with guanosine 5′-triphospate, a candidate substance and nitric oxide in a cell by introducing a polynucleotide expressing the probe and then the candidate substance into the cell. 
     
     
         13 . The method according to  claim 11 , wherein the probe coexists with guanosine 5′-triphospate, a candidate substance and nitric oxide in a cell by introducing a pair of polynucleotides respectively expressing a hybrid protein into the cell, wherein each of the hybrid protein comprises α- or β-subunits of soluble guanylate cyclase connecting with the cGMP-visualization probe. 
     
     
         14 . The method according to  claim 11 , wherein the probe coexists with guanosine 5′-triphospate, a candidate substance and nitric oxide in all cells of a non-human animal or its progeny by administering the candidate substance to the animal or its progeny, wherein the animal is established by introducing polynucleotide expressing the probe into a non-human totipotent cell and developing the cell to individual. 
     
     
         15 . The method according to  claim 11 , wherein the probe coexists with guanosine 5′-triphospate, a candidate substance and nitric oxide in all cells of a non-human animal or its progeny by administering the candidate substance to the animal or its progeny, wherein the animal is established by introducing a pair of polynucleotides respectively expressing a hybrid protein into a non-human totipotent cell and developing the cell to individual, and wherein each of the hybrid protein comprises α- or β-subunits of soluble guanylate cyclase connecting with the cGMP-visualization probe. 
     
     
         16 . A method for monitoring an intracellular change of nitric oxide concentration with a stimulation, which comprises introducing a polynucleotide expressing the probe of  claim 1  into a cell, stimulating the cell, and measuring signals before and after the stimulation. 
     
     
         17 . A method for monitoring an intracellular change of nitric oxide concentration with a stimulation, which comprises introducing a pair of polynucleotides respectively expressing a hybrid protein, stimulating the cell, and measuring signals before and after the stimulation, wherein each of the hybrid protein comprises α- or β-subunits of soluble guanylate cyclase connecting with the cGMP-visualization probe. 
     
     
         18 . A method for monitoring an intracellular change of nitric oxide concentration with a stimulation, which comprises stimulating a non-human animal or its progeny, and measuring signals before and after the stimulation, wherein the animal is established by introducing polynucleotide expressing the probe of  claim 1  into a non-human totipotent cell and developing the cell to individual. 
     
     
         19 . A method for monitoring an intracellular change of nitric oxide concentration with a stimulation, which comprises stimulating a non-human animal or its progeny, and measuring signals before and after the stimulation, wherein the animal is established by introducing a pair of polynucleotides respectively expressing a hybrid protein into a non-human totipotent cell and developing the cell to individual, and wherein each of the hybrid protein comprises α- or β-subunits of soluble guanylate cyclase connecting with the cGMP-visualization probe.