Long structured RNAs are useful biochemical and biological tools. They are usually prepared enzymatically, but this precludes their site-specific modification with functional groups for chemical biology studies. One solution is to perform solid-phase synthesis of multiple RNAs loaded with 5'-terminal phosphate groups, so that RNAs can be concatenated using template ligation reactions. However, there are currently no readily available reagents suitable for the incorporation of the phosphate group into long RNAs by solid-phase synthesis. Here we describe an easy-to-prepare phosphoramidite reagent suitable for the chemical introduction of 5'-terminal phosphate groups into long RNAs. The phosphate is protected by a dinitrobenzhydryl group that serves as an essential lipophilic group for the separation of oligonucleotide by-products. The phosphate is unmasked quantitatively by brief UV irradiation. We demonstrate the value of this reagent in the preparation of a library of long structured RNAs that are site-specifically modified with functional groups.
Keywords: RNA; dinitrobenzhydryl; phosphate; phosphoramidite; photolabile protecting group.
© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.