Complementary-addressed site-directed spin labeling of long natural RNAs.
Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires site directed spin labeling (SDSL) of biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient spin labeling and following structural EPR studies of long RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry site consisting of ≈330 nucleotides and having a complicated spatial structure. Application of pulsed double electron-electron resonance provided spin-spin distance distribution, which agrees well with the results of molecular dynamics (MD) calculations. Thus, novel SDSL approach in conjunction with EPR and MD allows structural studies of long natural RNAs with nanometer resolution and can be applied to systems of biological and biomedical significance.
Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia Novosibirsk State University, Novosibirsk 630090, Russia.
Elena S
ES
Babaylova
Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia Novosibirsk State University, Novosibirsk 630090, Russia.
Alexey A
AA
Malygin
Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia Novosibirsk State University, Novosibirsk 630090, Russia.
Alexander A
AA
Lomzov
Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia Novosibirsk State University, Novosibirsk 630090, Russia.
Dmitrii V
DV
Pyshnyi
Laboratory of Physical Chemistry, ETH Zurich, Zurich 8093, Switzerland.
Maxim
M
Yulikov
Laboratory of Physical Chemistry, ETH Zurich, Zurich 8093, Switzerland.
Gunnar
G
Jeschke
Novosibirsk State University, Novosibirsk 630090, Russia International Tomography Center SB RAS, Novosibirsk 630090, Russia.
Olesya A
OA
Krumkacheva
Novosibirsk State University, Novosibirsk 630090, Russia International Tomography Center SB RAS, Novosibirsk 630090, Russia mfedin@tomo.nsc.ru.
Matvey V
MV
Fedin
Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia Novosibirsk State University, Novosibirsk 630090, Russia karpova@niboch.nsc.ru.
Galina G
GG
Karpova
Novosibirsk State University, Novosibirsk 630090, Russia N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Novosibirsk 630090, Russia egbagryanskaya@nioch.nsc.ru.
Elena G
EG
Bagryanskaya
D000478:Alkylation; D001483:Base Sequence; D004578:Electron Spin Resonance Spectroscopy; D016174:Hepacivirus / Q000235:genetics*; D000067556:Internal Ribosome Entry Sites; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013113:Spin Labels; D013696:Temperature
D000067556:Internal Ribosome Entry Sites; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D013113:Spin Labels