Purification of oligonucleotides

Abstract

Methods and compounds useful for the purification of oligonucleotides and their analogs are provided wherein the oligonucleotides are contaminated with at least one oligonucleotide having at least one abasic site by the formation of imines at the aldehyde moiety of the abasic site and subsequent separation based on extractions, precipitations or chromatography.

What is claimed is:  
     
         1 . A method of purifying an oligonucleotide from a mixture, said mixture including said oligonucleotide and at least one contaminant comprising: 
 treating said mixture with an amino reagent reactive with said contaminant for a time and under conditions effective to form an imine linkage with each said contaminant; and    separating said oligonucleotide from said imine linked contaminants.    
     
     
         2 . The method of  claim 1  wherein said amino reagent is an amine, hydrazine, hydroxylamine, semicarbazide or thiosemicarbazide.  
     
     
         3 . The method of  claim 2  wherein said separation is based upon differences in solubility of said oligonucleotide and said imine linked contaminants in a selected solvent.  
     
     
         4 . The method of  claim 3  wherein said imine linked contaminants are more soluble in a selected solvent than said oligonucleotide.  
     
     
         5 . The method of  claim 3  wherein said imine linked contaminants are less soluble in a selected solvent than said oligonucleotide.  
     
     
         6 . The method of  claim 1  wherein said oligonucleotide is more soluble in a first solvent than said imine linked contaminant and said imine linked contaminant is more soluble in a second solvent than said oligonucleotide and said first and said second solvents are immiscible.  
     
     
         7 . The method of  claim 6  wherein said first solvent is water or an aqueous solution and said second solvent is an organic solvent.  
     
     
         8 . The method of  claim 7  wherein said organic solvent is benzene, diethyl ether, ethyl acetate, hexanes, pentane, chloroform, dichloromethane or carbon tetrachloride.  
     
     
         9 . The method of  claim 1  wherein said oligonucleotide is more soluble in a first solvent than said imine linked contaminant and said imine linked contaminant is more soluble in a second solvent than said oligonucleotide and said first and said second solvents are miscible.  
     
     
         10 . The method of  claim 9  wherein said first solvent is water or an aqueous solution.  
     
     
         11 . The method of  claim 9  wherein said second solvent is an organic solvent.  
     
     
         12 . The method of  claim 11  wherein said organic solvent is acetone, methanol, ethanol or isopropanol.  
     
     
         13 . The method of  claim 1  wherein said separating is effected by selectively precipitating the oligonucleotide or the imine linked contaminant.  
     
     
         14 . The method of  claim 13  wherein said separating is effected by selectively precipitating the oligonucleotide.  
     
     
         15 . The method of  claim 1  wherein said separating is effected by chromatography.  
     
     
         16 . The method of  claim 1  wherein said separating is effected by liquid-liquid extraction.  
     
     
         17 . The method of  claim 1  wherein said amino reagent further includes a surfactant.  
     
     
         18 . The method of  claim 17  wherein said surfactant is a non-ionic surfactant.  
     
     
         19 . The method of  claim 17  wherein said amino reagent further including said surfactant has the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 x is from 0 to 20,  
 y is from 0 to 5,  
 n is from 0 to 150; and  
 Z is —NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2  or —NHC(S)NHNH 2 .  
 
     
     
         20 . The method of  claim 19  wherein x is 8, y is 1 and n is 12.  
     
     
         21 . The method of  claim 1  wherein said amino reagent further includes an attachment to a polymeric support.  
     
     
         22 . The method of  claim 21  wherein said polymeric support is a solid phase polymeric support.  
     
     
         23 . The method of  claim 21  wherein said polymeric support is a hydroxylamine resin.  
     
     
         24 . The method of  claim 21  wherein said separating is effected by rinsing or washing said oligonucleotide from said imine linked contaminants bound to said polymeric support.  
     
     
         25 . The method of  claim 21  wherein said polymeric support is a liquid phase polymeric support.  
     
     
         26 . The method of  claim 25  wherein said liquid phase polymeric support is hydrophilic.  
     
     
         27 . The method of  claim 25  wherein said polymeric support is a polyvinyl alcohol, a polyethylene glycol (PEG), a cellulose, or a polyvinyl alcohol-poly(1-vinyl-2-pyrrolidinone).  
     
     
         28 . The method of  claim 25  wherein said amino reagent including said polymeric support is a polyethylene glycol (PEG) amine, polyethylene glycol (PEG) hydrazine, polyethylene glycol (PEG) hydroxylamine, polyethylene glycol (PEG) semicarbazide, polyethylene glycol (PEG) thiosemicarbazide.  
     
     
         29 . A method of purifying an oligonucleotide from a mixture, said mixture including said oligonucleotide and at least one contaminant, comprising: 
 treating said mixture with a compound of formula I:                          I    wherein:    x is from 0 to about 20,    y is from 0 to about 5,    n is from 0 to about 150,    Z is a reactive nitrogenous moiety capable of reacting with an aldehyde to form an imine;    for a time and under conditions effective to form an imine linkage with each contaminant; and    separating said oligonucleotide from said imine linked contaminants.    
     
     
         30 . The method of  claim 29  comprising treating said mixture with plurality of compounds of formula I wherein said compounds differ with respect to the value of n.  
     
     
         31 . The method of  claim 29  wherein n is from about 8 to about 16.  
     
     
         32 . The method of  claim 29  wherein n is 12.  
     
     
         33 . The method of  claim 29  wherein Z is —NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2  or —NHC(S)NHNH 2 .  
     
     
         34 . The method of  claim 29  wherein Z is —ONH 2 .  
     
     
         35 . The method of  claim 29  wherein Z is —ONH 2 , x is 8, y is 1 and [CH 3 (CH 2 ) x ] y — is para to said —(OCH 2 CH 2 ) n -Z.  
     
     
         36 . The method of  claim 35  wherein n is from about 8 to 16.  
     
     
         37 . The method of  claim 35  wherein n is 12.  
     
     
         38 . The method of  claim 29  wherein said separating is effected by liquid-liquid extraction.  
     
     
         39 . The method of  claim 29  wherein said separating is effected by selectively precipitating the oligonucleotide or the imine linked contaminant.  
     
     
         40 . The method of  claim 39  wherein said separating is effected by selectively precipitating the oligonucleotide.  
     
     
         41 . The method of  claim 29  wherein said separating is effected by chromatography.  
     
     
         42 . A method of modifying the solubility of an oligonucleotide comprising: 
 selecting an oligonucleotide having at least one abasic site;    treating said oligonucleotide with an amino reagent reactive with said oligonucleotide for a time and under conditions effective to form an imine linkage between said oligonucleotide and said amino reagent at said abasic site.    
     
     
         43 . The method of  claim 42  wherein said amino reagent is an amine, hydrazine, hydroxylamine, semicarbazide or thiosemicarbazide.  
     
     
         44 . The method of  claim 42  wherein said amino reagent further includes a surfactant.  
     
     
         45 . The method of  claim 44  wherein said surfactant is a non-ionic surfactant.  
     
     
         46 . The method of  claim 44  wherein said amino reagent further including said surfactant has the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 x is from 0 to 20,  
 y is from 0 to 5,  
 n is from 0 to 150; and  
 Z is —NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2  or —NHC(S)NHNH 2 .  
 
     
     
         47 . The method of  claim 46  wherein x is 8, y is 1 and n is 12 said.  
     
     
         48 . The method of  claim 42  wherein said amino reagent further includes an attachment to a polymeric support.  
     
     
         49 . The method of  claim 48  wherein said polymeric support is a solid phase polymeric support.  
     
     
         50 . The method of  claim 48  wherein said polymeric support is an hydroxylamine resin.  
     
     
         51 . The method of  claim 48  wherein said polymeric support is a liquid phase polymeric support.  
     
     
         52 . The method of  claim 51  wherein said liquid phase polymeric support is hydrophilic.  
     
     
         52 . The method of  claim 51  wherein said polymeric support is a polyvinyl alcohol, a polyethylene glycol (PEG), a cellulose, or a polyvinyl alcohol-poly(1-vinyl-2-pyrrolidinone).  
     
     
         53 . The method of  claim 51  wherein said amino reagent further included to a polymeric support is a polyethylene glycol (PEG) amine, polyethylene glycol (PEG) hydrazine, polyethylene glycol (PEG) hydroxylamine, polyethylene glycol (PEG) semicarbazide, polyethylene glycol (PEG) thiosemicarbazide.  
     
     
         54 . A compound of Formula I,  
       
         
           
           
               
               
           
         
         x is from 0 to 20,  
         y is from 0 to 5,  
         n is from 0 to 150; and  
         Z is —NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2  or —NHC(S)NHNH 2 .  
       
     
     
         55 . A composition comprising a plurality of compounds according to  claim 54  wherein said compounds differ with respect to the value of n; wherein said value of n is from 0 to about 150.  
     
     
         56 . The composition of  claim 54  wherein said value of n is from about 8 to about 16.  
     
     
         57 . The compound of  claim 54  wherein n is 12.  
     
     
         58 . The compound of  claim 54  wherein Z is —ONH 2 .  
     
     
         59 . The compound of  claim 54  wherein Z is —ONH 2 , x is 8, y is 1, [CH 3 (CH 2 ) x ] y  is para to said (OCH 2 CH 2 ) n -Z, and n is from about 0 to about 150.  
     
     
         60 . The compound of  claim 59  wherein n is from about 8 to 16.  
     
     
         61 . The compound of  claim 59  wherein n is 12.