NMR Spectroscopy of Large Functional RNAs: From Sample Preparation to Low-Gamma Detection

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Nuclear Magnetic Resonance (NMR) spectroscopy is crucial for RNA structural and biophysical studies.
• Current NMR applications for RNA are limited by RNA size and often require isotopic labeling.
• Advancements in NMR pulse sequences, particularly heteronuclear-detected experiments, offer new possibilities.

Purpose of the Study:

• To provide detailed protocols for preparing isotope-labeled RNA suitable for NMR spectroscopy.
• To present a chemo-enzymatic method for site-specific RNA modification.
• To detail the implementation of advanced heteronuclear-detected NMR experiments for RNA analysis.

Main Methods:

• In vitro transcription for milligram-scale production of isotope-labeled RNA.
• Chemo-enzymatic synthesis for introducing single modified nucleotides at specific RNA positions.
• Implementation of 13C-detected experiments for ribose and amino group assignment.
• Application of CN-spin filter heteronuclear single quantum coherence (HSQC) for imino groups.
• Utilizing 15N-detected band-selective excitation short transient transverse-relaxation-optimized spectroscopy (BEST-TROSY) experiments.

Main Results:

• Established protocols for preparing milligram quantities of isotope-labeled RNA via in vitro transcription and purification.
• Developed a chemo-enzymatic approach for site-specific RNA labeling.
• Detailed implementation of various 13C- and 15N-detected NMR experiments for comprehensive RNA characterization.
• Demonstrated methods for ribose assignment, amino group detection, and imino group analysis using advanced NMR techniques.

Conclusions:

• The provided protocols facilitate the preparation of isotope-labeled and site-specifically modified RNAs for NMR studies.
• Advanced heteronuclear-detected NMR experiments enable detailed structural and biophysical characterization of RNAs.
• These methods expand the scope and accessibility of NMR spectroscopy for RNA research.