Synthesis, deprotection, analysis & purification of RNA & ribozymes

Abstract

Method for purification and synthesis of RNA molecules and enzymatic RNA molecules in enzymatically active form.

1 . A process for chemically synthesizing, deprotecting, and purifying RNA having one or more chemical modifications, comprising: 
 a. contacting a phosphoramidite with S-alkyltetrazole under conditions for synthesizing said RNA;    b. contacting said RNA with an alkylamine under conditions for removing any exocyclic amine protecting groups or phosphate ester protecting groups;    c. contacting said RNA with triethylamine-hydrogen fluoride under conditions to remove any alkylsilyl protecting groups from said RNA;    d. loading said RNA onto an anion exchange high-performance liquid chromatography (HPLC) column;    e. eluting said RNA by passing a buffer through said column; and    f. collecting the eluate from said column and recovering said RNA from said eluate, under conditions which allow for the purification of said RNA.    
     
     
         2 . The process of  claim 1 , wherein said RNA is provided as a sodium, potassium, or magnesium salt.  
     
     
         3 . The process of  claim 1 , wherein said RNA is provided in desalted form.  
     
     
         4 . The process of  claim 1 , wherein said anion exchange column comprises resins that are either quaternary or tertiary amino derivatized stationary phases.  
     
     
         5 . The process of  claim 4 , wherein said resin is either silica-based or polystyrene based.  
     
     
         6 . The process of  claim 1 , wherein said RNA comprises a plurality of chemical modifications.  
     
     
         7 . The process of  claim 1 , wherein said chemical modification is a sugar modification.  
     
     
         8 . The process of  claim 1 , wherein said chemical modification is a base modification.  
     
     
         9 . The process of  claim 1 , wherein said chemical modification is a phosphate backbone modification.  
     
     
         10 . The process of  claim 7 , wherein said sugar modification is a 2′-O-methyl modification.  
     
     
         11 . The process of  claim 7 , wherein said sugar modification is a 2′-deoxy-2′-amino modification.  
     
     
         12 . The process of  claim 7 , wherein said sugar modification is a 2′-deoxy-2′-fluoro modification.  
     
     
         13 . The process of  claim 9 , wherein said phosphate backbone modification is a phosphorothioate modification.  
     
     
         14 . The process of  claim 1 , wherein said RNA is chemically synthesized using solid phase synthesis.  
     
     
         15 . The process of  claim 1 , wherein said S-alkyltetrazole is S-ethyltetrazole.  
     
     
         16 . The process of  claim 1 , wherein said alkylamine is methylamine.  
     
     
         17 . The process of  claim 1 , wherein said alkylamine is ethylamine.  
     
     
         18 . The process of  claim 1 , wherein said alkylamine is triethylamine.