Consolidated 3‑Fold Isotopic Lens for Probing RNAs.

Area of Science:

• Biochemistry
• Molecular Biology
• Spectroscopy

Background:

• Nuclear magnetic resonance (NMR) spectroscopy is crucial for studying RNA structure and dynamics.
• Scalar and dipolar couplings often complicate NMR analyses.
• Site-specific isotopic labeling has improved RNA studies by enabling controlled isotope incorporation.

Purpose of the Study:

• To synthesize and incorporate a novel [1'-13C, 2-19F, 7-15N] adenosine triphosphate (ATP) probe into ribosomal RNA (rRNA).
• To expand the utility of existing isotopic labeling methods for RNA analysis.
• To leverage 19F incorporation for monitoring binding interactions and studying RNA dynamics.

Main Methods:

• Site-specific isotopic labeling of adenosine triphosphate with 13C, 19F, and 15N isotopes.
• Incorporation of the labeled ATP into a 9 kDa rRNA molecule.
• Utilizing 13C and 15N probes for structural and dynamic characterization via NMR.
• Employing 19F NMR for monitoring binding events.
• Analyzing R1 and R1ρ relaxation rates using 15N7-adenosine chemical shielding anisotropy.

Main Results:

• Successful synthesis and incorporation of [1'-13C, 2-19F, 7-15N] ATP into rRNA.
• Demonstrated the utility of 13C and 15N probes for characterizing RNA structural dynamics.
• Showcased 19F as an effective tool for monitoring binding interactions.
• Enabled straightforward analysis of R1 and R1ρ relaxation rates.

Conclusions:

• The novel isotopic labeling strategy expands the capabilities of NMR spectroscopy for RNA research.
• The combined use of 13C, 15N, and 19F provides a powerful approach for detailed RNA structural and dynamic studies.
• This method facilitates the investigation of RNA-protein interactions and dynamics at a molecular level.