6 - Chemical RNA Synthesis (Including RNA with Unusual Constituents)

Abstract

This review discusses some protecting groups and coupling reagents used in RNA synthesis and describes methods for the synthesis of RNA derivatives. The chemical synthesis of RNA has been developed to the stage of automated large scale production. This provides substantial amounts of RNA needed for physicochemical and structural studies. The yield of oligoribonucleotides also depends on the efficiency of the deprotection and separation from the side products. A variety of combinations of protection methods for the 2-hydroxyl and 5-hydroxyl groups has been employed for RNA synthesis. Larger molecules such as tRNAs consisting of approximately 80 nucleotides have been obtained by solid-phase synthesis using the phosphoramidite approach. Practical sizes of RNA for physicochemical studies are still limited to between 10 and 20 nucleotides. Enzymatic synthesis of RNA by transcription or enzymatic ligation of oligoribonucleotides has been used for larger oligoribonucleotides to compensate for the limitations of RNA synthesis. The two approaches used are the solid-phase synthesis of RNA and the synthesis in solutions. The replacement of modified RNA fragments is another method of incorporating unusual nucleosides in defined positions, provided that methods for specific cleavage and joining of the RNA fragments are available.