Phosphorothioate-Based Site-Specific Labeling of Large RNAs for Structural and Dynamic Studies

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Molecular rulers like Pulsed Electron-Electron Double Resonance (PELDOR) spectroscopy, X-ray scattering interferometry (XSI), and single-molecule Förster resonance energy transfer (smFRET) are crucial for measuring distances in biomolecules.
• Site-specific labeling is essential for these techniques, but current methods for large RNAs using unnatural base pairs can cause structural perturbations and reduce measurement accuracy.

Purpose of the Study:

• To develop a novel site-specific labeling strategy for large RNAs that minimizes structural perturbation and enhances the accuracy of distance measurements.
• To introduce an α-thiophosphorylated variant of rTPT3TP (rTPT3αS) for labeling the RNA backbone.

Main Methods:

• Synthesis of the α-thiophosphorylated variant rTPT3αS.
• Post-transcriptional site-specific labeling of large RNAs at the α-phosphate backbone using maleimide-modified probes.
• Application of PELDOR, XSI, and smFRET to measure pairwise distances.

Main Results:

• The new labeling strategy using rTPT3αS enables site-specific modification of the RNA backbone.
• Labeling via the α-phosphate backbone resulted in narrower distance distributions compared to labeling at the unnatural base pair (UBP).
• This indicates improved accuracy in distance measurements for structural and dynamic studies.

Conclusions:

• The developed rTPT3αS labeling strategy offers a more precise approach for structural and dynamic studies of large RNAs.
• This method overcomes limitations of previous labeling techniques, enhancing the utility of molecular rulers.
• It provides a new avenue for investigating the structure and dynamics of complex RNA molecules.