Towards novel efficient and stable nuclear import signals: synthesis and properties of trimethylguanosine cap analogs modified within the 5′,5′-triphosphate bridge

A trimethylguanosine (TMG) cap is present at the 5′ end of several small nuclear and nucleolar RNAs. Recently, it has been reported that the TMG cap is a potential nuclear import signal for nucleus-targeting therapeutic nucleic acids and proteins. The import is mediated by recognition of the TMG cap by the snRNA transporting protein, snurportin1. This work describes the synthesis and properties of a series of dinucleotide TMG cap (m32,2,7GpppG) analogs modified in the 5′,5′-triphosphate bridge as tools to study TMG cap-dependent biological processes. The bridge was altered at different positions by introducing either bridging (imidodiphosphate, O to NH and methylenebisphosphonate, O to CH2) or non-bridging (phosphorothioate, O to S and boranophosphate, O to BH3) modifications, or by elongation to tetraphosphate. The stability of novel analogs in blood serum was studied to reveal that the α,β-bridging O to NH substitution (m32,2,7GppNHpG) confers the highest resistance. Short RNAs capped with analogs containing α,β-bridging (m32,2,7GppNHpG) or β-non-bridging (m32,2,7GppSpG D2) modifications were resistant to decapping pyrophosphatase, hNudt16. Preliminary studies on binding by human snurportin1 revealed that both O to NH and O to S substitutions support this binding. Due to favorable properties in all three assays, m32,2,7GppNHpG was selected as a promising candidate for further studies on the efficiency of the TMG cap as a nuclear import signal.