Novel solid-phase synthesis of branched oligoribonucleotides, including a substrate for the RNA debranching enzyme

Abstract

An effective new route for synthesizing branched oligoribonucleotides in the solid phase in the 5′ to 3′ direction has been developed. This required the synthesis of reversed monomers, viz. protected nucleoside 5′-phosphoramidites bearing 2′-O-Fpmp and (3′-O-pixy) protecting groups as well as special branch-point monomers, viz. protected nucleoside 5′-phosphoramidites bearing either 2′, 3′-O-dipixyl protection in the case of adenosine, cytidine and uridine, or 2′,3′-O-dilaevulinyl protection in the case of guanosine. These monomers are assembled on commercial synthesizers into branched oligoribonucleotides in high yield, the crude products are readily purified by reversedphase HPLC whilst still partially protected, and the fully deprotected products are conveniently analysed by electrospray mass spectrometry. Moreover, the branched oligoribonucleotides can be recognised and cleaved by a specific 2′–5′ phosphodiesterase present in mammalian cell nuclei. We expect that this will prove valuable for future biochemical and biological studies on the properties of branched RNA molecules and the protein factors and enzymes that interact with branched RNA substrates.