Methods for detecting and quantifying binding and inhibition of binding of species to nucleic acids

Abstract

The invention features methods for detecting and quantifying the binding or inhibition of binding of species to biopolymers, e.g., nucleic acids. The invention is based on the use of probes that have magnetic relaxation properties that are affected by the presence of paramagnetic metal ions, e.g., Mn 2+ . Any class of biopolymer that binds metal ions at its active site or uses metal ion cofactors can be studied using these methods.

1 . A method of detecting the binding of a species to a biopolymer other than a protein, said method comprising the steps of: 
 (a) measuring a magnetic relaxation property of a probe in a first solution using nuclear magnetic resonance spectroscopy, wherein said first solution comprises said biopolymer, a first concentration of said species, a paramagnetic metal ion, and said probe;    (b) measuring the magnetic relaxation property of said probe in a second solution using nuclear magnetic resonance spectroscopy, wherein said second solution comprises said biopolymer, a second concentration of said species, a paramagnetic metal ion, and said probe; and    (c) comparing the relaxation property measured in step (a) with the relaxation property measured in step (b), wherein a difference in said relaxation properties indicates the binding of said species to said biopolymer.    
     
     
         2 . The method of  claim 1 , wherein, in step (c), said magnetic relaxation properties of said probe are correlated with said first and second concentrations of said species, thereby quantifying the binding of said species to said biopolymer.  
     
     
         3 . The method of  claim 1 , wherein said first concentration is 0.0 μM.  
     
     
         4 .- 5 . (canceled)  
     
     
         6 . The method of  claim 1 , wherein said species is a candidate therapeutic agent.  
     
     
         7 .- 9 . (canceled)  
     
     
         10 . The method of  claim 1 , wherein said paramagnetic metal ion is bound to said biopolymer.  
     
     
         11 . The method of  claim 1 , wherein said probe comprises an X—H bond, wherein X is an NMR-active nucleus.  
     
     
         12 . The method of  claim 11 , wherein X is  31 P.  
     
     
         13 . The method of  claim 12 , wherein said probe is methylphosphite, ethylphosphite, or ethylphosphonite.  
     
     
         14 . The method of  claim 11 , wherein said magnetic relaxation property of X is indirectly detected using X edited,  1 H detected NMR spectroscopy.  
     
     
         15 . (canceled)  
     
     
         16 . The method of  claim 1 , wherein said magnetic relaxation property is the T 2  relaxation of a nucleus of said probe.  
     
     
         17 . The method of  claim 1 , wherein said biopolymer comprises a nucleic acid.  
     
     
         18 . The method of  claim 1 , wherein said biopolymer comprises a polypeptide.  
     
     
         19 . The method of  claim 1 , wherein said biopolymer comprises a polysaccharide.  
     
     
         20 . A method of detecting the binding of a paramagnetic metal ion to a biopolymer other than a protein, said method comprising the steps of: 
 (a) measuring a magnetic relaxation property of a probe in a first solution using nuclear magnetic resonance spectroscopy, wherein said first solution comprises a first concentration of said paramagnetic metal ion, said biopolymer, and said probe;    (b) measuring the magnetic relaxation property of said probe in a second solution using nuclear magnetic resonance spectroscopy, wherein said second solution comprises a second concentration of said paramagnetic metal ion, said biopolymer, and said probe; and    (c) comparing the relaxation property measured in step (a) with the relaxation property measured in step (b), wherein a difference in said relaxation properties indicates the binding of said paramagnetic metal ion to said biopolymer.    
     
     
         21 . The method of  claim 20 , wherein, in step (c), said magnetic relaxation properties of said probe are correlated with said first and second concentrations of said nucleic acid, thereby quantifying the binding of said paramagnetic metal ion to said nucleic acid.  
     
     
         22 . The method of  claim 20 , wherein said first concentration is 0.0 μM.  
     
     
         23 . (canceled)  
     
     
         24 . The method of  claim 1 , wherein said probe comprises an X—H bond, wherein X is an NMR-active nucleus.  
     
     
         25 . The method of  claim 18 , wherein X is  31 P.  
     
     
         26 . The method of  claim 25 , wherein said probe is methylphosphite, ethylphosphite, or ethylphosphonite.  
     
     
         27 . A method of determining the effect of a first species on the binding of a second species to a biopolymer other than a protein, said method comprising the steps of: 
 (a) measuring a magnetic relaxation property of a probe in a first solution using nuclear magnetic resonance spectroscopy, wherein said first solution comprises a first concentration of said first species, said biopolymer, said second species that binds to said biopolymer, a paramagnetic metal ion, and a probe;    (b) measuring the magnetic relaxation property of said probe in a second solution using nuclear magnetic resonance spectroscopy, wherein said second solution comprises a second concentration of said first species, said biopolymer, said second species, said paramagnetic metal ion, and said probe; and    (c) comparing the relaxation property measured in step (a) with the relaxation property measured in step (b), wherein a difference in said relaxation properties indicates the inhibition of the binding of said second species to said biopolymer.    
     
     
         28 . The method of  claim 27 , wherein, in step (c), said magnetic relaxation properties of said probe are correlated with said first and second concentrations of said first species, thereby quantifying the effect of said first species on the binding of said second species to said biopolymer.  
     
     
         29 . The method of  claim 27 , wherein said first concentration is 0.0 μM.  
     
     
         30 .- 31 . (canceled)  
     
     
         32 . The method of  claim 27 , wherein said first species or said second species is a candidate therapeutic agent.  
     
     
         33 .- 35 . (canceled)  
     
     
         36 . The method of  claim 27 , wherein said paramagnetic metal ion is bound to said biopolymer.  
     
     
         37 . The method of  claim 27 , wherein said probe comprises an X—H bond, wherein X is an NMR-active nucleus.  
     
     
         38 . The method of  claim 27 , wherein X is  31 P.  
     
     
         39 . The method of  claim 38 , wherein said probe is methylphosphite, ethylphosphite, or ethylphosphonite.  
     
     
         40 . A method of detecting the availability of a paramagnetic metal ion in a solution comprising a biopolymer other than a protein, said method comprising the steps of: 
 (a) providing a solution comprising said biopolymer, a probe, and said paramagnetic metal ion and    (b) measuring a magnetic relaxation property of said probe in said solution using nuclear magnetic resonance spectroscopy, wherein the magnitude of said magnetic property is indicative of the availability of said paramagnetic ion in said solution.    
     
     
         41 . The method of  claim 40 , wherein said paramagnetic metal ion is bound to said biopolymer.