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Estimation of Telomeric Repeat-containing RNA from DNA/RNA Hybrid Complexes
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RNA denaturation underlies circular RNA separation.

Yanyi Jiang1, Jørgen Kjems1,2

  • 1Interdisciplinary Nanoscience Center (iNANO), Aarhus University, 8000 Aarhus, Denmark.

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|November 20, 2025
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Summary
This summary is machine-generated.

Purifying circular RNAs (circRNAs) for therapeutics is challenging. This study shows that RNA denaturation is key for separating circRNAs from linear RNA using HPLC-SEC, improving purity for RNA therapeutics.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • RNA Therapeutics

Background:

  • Circular RNAs (circRNAs) are promising for RNA therapeutics due to stability and low immunogenicity.
  • Effective purification of synthesized circRNAs from linear RNA byproducts remains a significant challenge.
  • Current methods struggle with efficient separation, impacting therapeutic development.

Purpose of the Study:

  • To systematically evaluate gel electrophoresis and HPLC-SEC for circRNA purification.
  • To identify critical factors affecting circRNA separation efficiency.
  • To optimize purification methods for scalable circRNA therapeutic production.

Main Methods:

  • Comparative analysis of gel electrophoresis and HPLC-SEC for circRNA separation.
  • Investigation of ligase- and ribozyme-based synthesis strategies' impact on purification.
  • Assessment of RNA denaturation and magnesium ion concentration effects on HPLC-SEC performance.

Main Results:

  • Both gel electrophoresis and HPLC-SEC require RNA denaturation for effective circRNA separation.
  • Magnesium ions significantly hinder circRNA separation in HPLC-SEC, even in trace amounts.
  • Optimized denaturing HPLC-SEC allows direct purification from crude enzymatic reactions, simplifying the process.

Conclusions:

  • RNA denaturation is a critical parameter for successful circRNA purification using chromatography.
  • Optimized HPLC-SEC protocols can enhance the purity and scalability of circRNA therapeutics.
  • Mechanistic insights into circRNA separation advance the field of RNA-based drug development.