Application of NMR to Large RNAs

• Methods for determining large RNA structures (> 50 nucleotides). • Methods for probing larger RNAs (> 300 nucleotides). • Strengths and limitations of NMR approaches. • Methods for preparing and purifying RNAs with specific isotope incorporation. • Future directions for large RNA NMR. 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, 1 H- 13 C and 1 H- 15 N 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 1 H- 13 C dipolar coupling and difficulties obtaining and assigning 1 H- 15 N 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.