Alternative probe chemistries for single-molecule analysis of long noncoding RNA

Area of Science:

• Molecular Biology
• Biophysics
• Genomics

Background:

• Single-molecule microscopy is vital for RNA studies.
• Analyzing complex structures like long non-coding RNA (lncRNA) using methods like single-molecule kinetic analysis of RNA transient structure (SiM-KARTS) presents challenges.
• Optimization of experimental variables for SiM-KARTS on lncRNA is underexplored.

Purpose of the Study:

• To investigate the impact of alternative probe backbone chemistries on SiM-KARTS analysis of lncRNA.
• To establish design principles for applying SiM-KARTS to complex RNA targets.

Main Methods:

• Utilized SiM-KARTS, thermal denaturation, and circular dichroism spectroscopy.
• Analyzed binding behaviors of DNA probes with locked nucleic acid (LNA) and morpholino backbones.
• Employed a model lncRNA segment to control target sequence accessibility.

Main Results:

• Optimized probe backbone chemistry enables precise distinction between RNA structures and fine-tunes binding stability.
• Locked nucleic acid (LNA) probes demonstrated high sensitivity to RNA structure in binding and unbinding kinetics.
• Holistic analysis of binding and unbinding rates using LNA probes allowed accurate classification of different RNA structures.

Conclusions:

• Probe backbone chemistry is a critical variable for optimizing SiM-KARTS analysis of lncRNA.
• LNA probes provide a robust tool for high-accuracy structural classification of complex RNAs.
• This study offers design guidelines for extending SiM-KARTS to challenging RNA targets like lncRNA.