Evaluation of ¹⁵N-detected H-N correlation experiments on increasingly large RNAs

Recently, ¹⁵N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a ¹⁵N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three ¹⁵N-detected H-N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established ¹⁵N-detected BEST-TROSY experiment, which proves to be the most sensitive ¹⁵N-detected H-N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the ¹⁵N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the ¹H- than in the ¹⁵N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that ¹⁵N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.