Abstract
The N(1)-methyladenosine (m(1)A) epigenetic modification exists in many RNAs and is related to many human diseases. Chemically synthesized RNAs containing the modification are required for projects aimed at studying biological processes, mechanisms, and pathogenesis related to m(1)A. Existing methods for the synthesis of m(1)A containing RNAs use tetrabutylammonium fluoride (TBAF) for the deprotection of the 2'-silyl protecting groups. Since TBAF is nonvolatile, and is relatively non-polar, its use in the desilylation of RNA requires repeated desalting, which is tedious and gives low yields. Here we report the use of the volatile and neat triethylamine hydrogen fluoride (TEA-HF) for desilylation of m(1)A RNA synthesis. We found that the method is much simpler, and-in our hands-give significantly higher yield of RNA. Two major concerns for m(1)A RNA synthesis are depurination and Dimroth rearrangement. HPLC and MALDI MS of the RNA indicated that depurination is not a problem for the new method. The absence of Dimroth rearrangement is proven by RNA digestion followed by HPLC analysis of the nucleosides.