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Alternative probe chemistries for single-molecule analysis of long non-coding RNA
Kalika R Pai1, Aimee M Martin2, Julia R Widom1
1Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR 97403.
Biorxiv : the Preprint Server for Biology
|January 23, 2026
Summary
Optimizing probe chemistry enhances single-molecule kinetic analysis of RNA transient structure (SiM-KARTS) for complex long noncoding RNAs (lncRNAs). This allows precise structural distinction and tunable binding stability for lncRNA research.
Area of Science:
- Molecular Biology
- Biophysics
- RNA Biology
Background:
- Single-molecule microscopy is crucial for RNA structure and dynamics.
- Studying long noncoding RNAs (lncRNAs) presents challenges due to their size and complexity.
- Single-molecule kinetic analysis of RNA transient structure (SiM-KARTS) shows promise for lncRNA analysis.
Purpose of the Study:
- To optimize SiM-KARTS for complex systems like lncRNA.
- To investigate the impact of probe backbone chemistry on SiM-KARTS performance.
- To establish design principles for applying SiM-KARTS to lncRNAs.
Main Methods:
- Single-molecule kinetic analysis of RNA transient structure (SiM-KARTS).
- Thermal denaturation experiments.
- Circular dichroism spectroscopy.
- Analysis of lncRNA model systems with modified oligonucleotide probes (DNA with LNA, morpholinos).
Main Results:
- Optimized probe backbone chemistry allows for precise discrimination between different RNA structures.
- Alternative probe chemistries enable fine-tuning of binding stability without significant structural impact.
- Demonstrated successful adaptation of SiM-KARTS for lncRNA analysis.
Conclusions:
- Probe backbone chemistry is a critical variable for optimizing SiM-KARTS in complex RNA systems.
- Modified oligonucleotide probes offer enhanced specificity and stability for lncRNA structural studies.
- Provides foundational principles for applying SiM-KARTS to diverse and complex RNA targets.
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