Deleterious effects of carbon-carbon dipolar coupling on RNA NMR dynamics

Many regulatory RNAs undergo dynamic exchanges that are crucial for their biological functions and NMR spectroscopy is a versatile tool for monitoring dynamic motions of biomolecules. Meaningful information on biomolecular dynamics requires an accurate measurement of relaxation parameters such as longitudinal (R₁) rates, transverse (R₂) rates and heteronuclear Overhauser effect (hNOE). However, earlier studies have shown that the large ¹³C-¹³C interactions complicate analysis of the carbon relaxation parameters. To investigate the effect of ¹³C-¹³C interactions on RNA dynamic studies, we performed relaxation measurements on various RNA samples with different labeling patterns and compared these measurements with the computational simulations. For uniformly labeled samples, contributions of the neighboring carbon to R₁ measurements were observed. These contributions increased with increasing magnetic field and overall correlation time ([Formula: see text]) for R₁ rates, necessitating more careful analysis for uniformly labeled large RNAs. In addition, the hNOE measurements were also affected by the adjacent carbon nuclei. Unlike R₁ rates, R_1ρ rates showed relatively good agreement between uniformly- and site-selectively labeled samples, suggesting no dramatic effect from their attached carbon, in agreement with previous observations. Overall, having more accurate rate measurements avoids complex analysis and will be a key for interpreting ¹³C relaxation rates for molecular motion that can provide valuable insights into cellular molecular recognition events.