Bioreactive agents

Abstract

This invention relates to agents and conjugates that can be used to detect and isolate target components from complex mixtures such as nucleic acids from biological samples, cells from bodily fluids, and nascent proteins from translation reactions. Agents comprise a detectable moiety bound to a photoreactive moiety. Conjugates comprise agents coupled to substrates by covalent bounds which can be selectively cleaved with the administration of electromagnetic radiation. Targets substances labeled with detectable molecules can be easily identified and separated from a heterologous mixture of substances. Exposure of the conjugate to radiation releases the target in a functional form and completely unaltered. Using photocleavable molecular precursors as the conjugates, label can be incorporated into macromolecules, the nascent macromolecules isolated and the label completely removed. The invention also relates to targets isolated with these conjugates which may be useful as pharmaceutical agents or compositions that can be administered to humans and other mammals. Useful compositions include biological agents such as nucleic acids, proteins, lipids and cytokines. Conjugates can also be used to monitor the pathway and half-life of pharmaceutical composition in vivo and for diagnostic, therapeutic and prophylactic purposes. The invention also relates to kits comprised of agents and conjugates that can be used for the detection of diseases, disorders and nearly any individual substance in a complex background of substances.

1 . A conjugate, comprising a substrate covalently coupled to a detectable moiety through a cleavable linker, said substrate selected from the group consisting of nucleosides and nucleotides, said detectable moiety having the property of fluorescence. 
     
     
         2 . The conjugate of  claim 1 , wherein said substrate is a nucleotide selected from the group consisting of NTP, dNTP and ddNTP. 
     
     
         3 . The conjugate of  claim 2 , wherein said nucleotide is part of an oligonucleotide. 
     
     
         4 . The conjugate of  claim 1 , wherein said detectable moiety comprises coumarin. 
     
     
         5 . The conjugate of  claim 1 , wherein said detectable moiety is selected from the group consisting of rhodamines and fluoresceins. 
     
     
         6 . A method, comprising:
 a) providing i) nucleic acid, ii) the conjugate of  claim 1 , and iii) a polymerase; and   b) mixing said nucleic acid and said polymerase in the presence of said conjugate under conditions such that said conjugate is incorporated into said nucleic acid to produce labeled nucleic acid.   
     
     
         7 . The method of  claim 6 , wherein said nucleic acid is RNA. 
     
     
         8 . The method of  claim 6 , wherein said nucleic acid is DNA.