Design, Synthesis, and Properties of Phosphoramidate 2',5'-Linked Branched RNA: Toward the Rational Design of Inhibitors of the RNA Lariat Debranching Enzyme.

Two RNA fragments linked by means of a 2′,5′ phosphodiester bridge (2′ hydroxyl of one fragment connected to the 5′ hydroxyl of the other) constitute a class of nucleic acids known as 2′-5′ branched RNAs (bRNAs). In this report we show that bRNA analogues containing 2′-5′ phosphoramidate linkages (bN-RNAs) inhibit the lariat debranching enzyme, a 2′,5′-phosphodiesterase that has recently been implicated in neurodegenerative diseases associated with aging. bN-RNAs were efficiently generated using automated solid-phase synthesis and suitably protected branchpoint building blocks. Two orthogonally removable groups, namely the 4-monomethoxytrityl (MMTr) group and the fluorenylmethyl-oxycarbonyl (Fmoc) groups, were evaluated as protecting groups of the 2′ amino functionality. The 2′-N-Fmoc methodology was found to successfully produce bN-RNAs on solid-phase oligonucleotide synthesis. The synthesized bN-RNAs resisted hydrolysis by the lariat debranching enzyme (Dbr1) and, in addition, were shown to attenuate th...