Application of NMR to Large RNAs

Heteronuclear NMR methodologies developed over the past 40 years have enabled atomic level insights into the solution-state structure and dynamics of proteins of ever-increasing size, some as large as 1 MDa. Unfortunately, ¹H-¹³C and ¹H-¹⁵N correlated methods foundational for studies of proteins have been less useful when applied to larger RNAs (>50 nucleotides; ∼17 kDa) due primarily to adverse relaxation effects caused by strong ¹H-¹³C dipolar coupling and difficulties obtaining and assigning ¹H-¹⁵N correlated spectra for exchangeable protons. Recently, alternative homo- and heteronuclear NMR approaches have been developed that involve nucleotide- and sequence-specific isotopic labeling. These methods have opened the door to structural probing of substantially larger RNAs (>700 nucleotides; ∼242 kDa). We herein review the applications, strengths, limitations, and exciting potential of these new approaches.