A versatile approach for site-directed spin labeling and structural EPR studies of RNAs.
Site-directed spin labeling (SDSL) is widely applied for structural studies of biopolymers by electron paramagnetic resonance (EPR). However, SDSL of long RNA sequences still remains a challenging task. Here, we propose a novel SDSL approach potentially suitable for long natural RNAs, which is based on the attachment of a linker containing an aliphatic amino group to the target nucleotide residue followed by selective coupling of a spin label to this amino group. Such a linker can be attached to the desired RNA residue via a sequence-specific reaction with the derivatives of oligodeoxyribonucleotides. To verify this approach, we applied it to model RNA duplex with known structure and expected distance between corresponding residues. A new 2,5-bis(spirocyclohexane)-substituted spin label with advanced stability and relaxation properties has been used, and the distance distribution measured using Q-band (34 GHz) pulsed double electron-electron resonance corresponds well to the expected one. We have additionally validated the obtained results by studying a similar RNA duplex, where the linker with the aliphatic amino group was introduced via solid-phase synthesis. Although this novel SDSL approach does not provide an advantage in precision of molecular distance measurements, we believe that its applicability to long RNAs is a crucial benefit for future structural studies using pulse EPR.
Institute of Chemical Biology and Fundamental Medicine SB RAS, pr. Lavrentjeva 8, Novosibirsk, 630090, Russia.
Elena S
ES
Babaylova
Anton V
AV
Ivanov
Alexey A
AA
Malygin
Maria A
MA
Vorobjeva
Alia G
AG
Venyaminova
Yuliya F
YF
Polienko
Igor A
IA
Kirilyuk
Olesya A
OA
Krumkacheva
Matvey V
MV
Fedin
Galina G
GG
Karpova
Elena G
EG
Bagryanskaya
D000478:Alkylation; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004583:Electrons; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D013113:Spin Labels
D018076:DNA, Complementary; D013113:Spin Labels; D012313:RNA