Relaxation Optimized Heteronuclear Experiments for Extending the Size Limit of RNA Nuclear Magnetic Resonance

Area of Science:

• Biophysics
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Heteronuclear correlation experiments are crucial for NMR analysis of biomolecules but are challenging for large RNAs (>60 nucleotides).
• Existing methods using 1H-13C correlations are impractical for large RNAs due to rapid dipolar relaxation, limiting structural and dynamic studies.
• Nuclear magnetic resonance (NMR) is a powerful tool for molecular structure determination, but its application to large RNAs has been hindered by technical limitations.

Purpose of the Study:

• To develop and validate a novel NMR approach for atom-specific characterization of large RNAs.
• To overcome the limitations of proton-carbon (1H-13C) correlations in large RNA molecules.
• To extend heteronuclear NMR techniques, routinely used for proteins, to the study of large RNAs.

Main Methods:

• A deuterium (2H)-enhanced, 1H-15N correlation NMR approach was developed.
• Ribose perdeuteration was employed to reduce proton transverse relaxation rates by approximately 20-fold.
• Focus was placed on H8-N9 correlation spectra and retention of C1' protons for chemical shift assignment and complementary correlations.

Main Results:

• The 2H-enhanced method enables efficient magnetization transfer via two-bond 1H-15N couplings in large RNAs.
• Atom-specific NMR characterization, including chemical shift assignments and 15N-edited nuclear Overhauser effects, was achieved for a 232-nucleotide RNA.
• High-resolution, sensitive spectra allowed for the measurement of 1H-15N residual dipolar couplings, facilitating structural analysis.

Conclusions:

• This novel NMR strategy significantly expands the applicability of heteronuclear NMR methods to large RNAs (up to 78 kDa).
• The technique provides unprecedented atom-specific insights into the structure and dynamics of large ribonucleic acid molecules.
• This advancement opens new avenues for detailed structural investigations of complex RNA systems previously inaccessible by NMR.