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

• Published in: Organic & biomolecular chemistry 2014-12, Vol.12 (45), p.9184-9199
• Main Authors: Zytek, Malgorzata, Kowalska, Joanna, Lukaszewicz, Maciej, Wojtczak, Blazej A, Zuberek, Joanna, Ferenc-Mrozek, Aleksandra, Darzynkiewicz, Edward, Niedzwiecka, Anna, Jemielity, Jacek
• Format: Article
• Language: English
• Subjects: Active Transport, Cell Nucleus; Guanosine - chemistry; Humans; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Molecular Structure; Polyphosphates - chemistry; Protein Binding; Pyrophosphatases - metabolism; Receptors, Cytoplasmic and Nuclear - metabolism; RNA Cap Analogs - chemical synthesis; RNA Cap Analogs - chemistry; RNA Cap-Binding Proteins - metabolism; Signal Transduction
• ISSN: 1477-0520; 1477-0539
• DOI: 10.1039/c4ob01579g

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 (m3(2,2,7)GpppG) 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 (m3(2,2,7)GppNHpG) confers the highest resistance. Short RNAs capped with analogs containing α,β-bridging (m3(2,2,7)GppNHpG) or β-non-bridging (m3(2,2,7)GppSpG 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, m3(2,2,7)GppNHpG was selected as a promising candidate for further studies on the efficiency of the TMG cap as a nuclear import signal.