New RNA labeling methods offer dramatic sensitivity enhancements in 2H NMR relaxation spectra.
A new labeling strategy is presented that greatly facilitates the measurement of 2H spin relaxation rates in RNA molecules as a probe of pico- to nanosecond time scale dynamics. In this labeling scheme the sugar positions are uniformly 13C-labeled, with position 2' protonated and all other sites on the sugar deuterated. Pulse sequences are presented for measurement of 2H R1 and R2 relaxation rates at positions 1', 3', and 4' with sensitivity gains that are on the order of 5-fold relative to previous methods that employed random fractional deuteration. The improved sensitivity is transformative and facilitates the study of motion in moderately sized RNA molecules with good sensitivity. The utility of the approach is demonstrated with an application to HIV-2 TAR, where the site-specific measures of molecular dynamics at sugar positions obtained here complement previous studies of dynamics at aromatic sites in the molecule.
Vallurupalli
Pramodh
P
Department of Medical Genetics, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S1A8, Canada.
Scott
Lincoln
L
Hennig
Mirko
M
Williamson
James R
JR
Kay
Lewis E
LE
D002247:Carbon Isotopes; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*
D002247:Carbon Isotopes; D012313:RNA