Methods of functionalization and reagents used in such methods using an aza-isatoic anhydride or a derivative thereof, biological molecules thus treated and kits

Abstract

A method of functionalizing RNA in a liquid sample includes reacting an anhydride function of a binding molecule with at least one RNA molecule at one or more hydroxyl groups in any of position 2′ of a ribose of any nucleotide of the RNA molecule, position 2′ of the ribose of the terminal nucleotide at the 3′ end of the RNA molecule, or position 3′ of the ribose of the terminal nucleotide at the 3′ end of the RNA molecule, to obtain functionalized RNA. The binding molecule includes an aza-isatoic anhydride or derivative thereof linked to a group of interest by a linkage, and an aza-anthranilate is formed from reacting the anhydride function of the binding molecule with a hydroxyl group of the RNA molecule so as to join the RNA molecule to the group of interest. The group of interest is a marker, labelling precursor, or ligand.

1 . A method of functionalizing RNA in a liquid sample, comprising:
 reacting an anhydride function of a binding molecule with at least one RNA molecule at one or more hydroxyl groups in any of (i) position 2′ of a ribose of any nucleotide of the RNA molecule, (ii) position 2′ of the ribose of the terminal nucleotide at the 3′ end of the RNA molecule, or (iii) position 3′ of the ribose of the terminal nucleotide at the 3′ end of the RNA molecule, to obtain functionalized RNA, wherein:   the binding molecule comprises an aza-isatoic anhydride or derivative thereof linked to a group of interest by a linkage;   an aza-anthranilate is formed from reacting the anhydride function of the binding molecule with a hydroxyl group of the RNA molecule so as to join the RNA molecule to the group of interest; and   the group of interest is a marker, a labelling precursor, or a ligand.   
     
     
         2 . The method according to  claim 1 , wherein the binding molecule is immobilized on a solid support. 
     
     
         3 . The method according to  claim 1 , wherein the linkage is a covalent bond or an optionally substituted carbon group having one or more carbon atoms and optionally containing an aromatic structure and/or heteroatom. 
     
     
         4 . The method according to  claim 1 , wherein the linkage comprises a bond or function capable of being cleaved in a physicochemical, photochemical, thermal, enzymatic, and/or chemical manner. 
     
     
         5 . The method according to  claim 1 , wherein the group of interest is the marker or labelling precursor. 
     
     
         6 . The method according to  claim 1 , wherein the aza-isatoic anhydride or derivative thereof has an intrinsic fluorescence and the group of interest is a marker having an intrinsic fluorescence different from that of the aza-isatoic anhydride or derivative thereof. 
     
     
         7 . The method according to  claim 1 , wherein the aza-isatoic anhydride or derivative thereof has an intrinsic fluorescence and the group of interest does not have an intrinsic fluorescence. 
     
     
         8 . The method according to  claim 1 , wherein the group of interest is a ligand complementary to an anti-ligand and the method further comprises:
 capturing the functionalized RNA by binding the ligand with the anti-ligand.   
     
     
         9 . The method according to  claim 8 , further comprising removing the captured RNA to obtain a DNA-enriched sample. 
     
     
         10 . The method according to  claim 1 , wherein the anhydride function of the binding molecule is reacted with the RNA molecule at room temperature. 
     
     
         11 . The method according to  claim 1 , further comprising hydrolysing the terminal monophosphate group in position 3′ of the RNA molecule. 
     
     
         12 . A method of separating RNA relative to other biological constituents including DNA, comprising:
 obtaining functionalized RNA by performing the method according to  claim 1  in a biological sample containing RNA and DNA wherein the group of interest is immobilized on a solid support that is able to be separated from the biological sample; and   separating the functionalized RNA from the biological sample.   
     
     
         13 . The method according to  claim 12 , wherein the functionalized RNA is obtained by performing the method at room temperature.