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Generation of precise and accurate engineered circRNAs using enzymatic ligation.

Amrita Singh1, Adela Dujsikova1, Noah Mueller1

  • 1Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06519, United States.

Nucleic Acids Research
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Summary

Engineered circular RNAs (circRNAs) offer enhanced stability over linear messenger RNA (mRNA) therapeutics. New methods ensure precise, homogenous circRNA production for reliable therapeutic and research applications.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • RNA Therapeutics

Background:

  • Linear messenger RNA (mRNA) therapeutics show promise but are limited by in vivo degradation.
  • Engineered circular RNAs (circRNAs) offer greater stability and are valuable for therapeutics and functional studies.
  • Precise and homogenous circRNA production is crucial for accurate biological interpretation.

Purpose of the Study:

  • To develop and optimize methods for generating precise, high-fidelity engineered circRNAs.
  • To overcome limitations of linear RNA degradation in therapeutic applications.
  • To provide a reliable method for producing homogenous circRNAs for research.

Main Methods:

  • Enzymatic ligation of linear RNAs was used instead of autocatalytic splicing to minimize extraneous nucleotides.
  • A permuted DNA transcription template with internal guanosines and a modified reverse primer was designed.
  • GMP-primed in vitro transcription, T4 RNA ligase 2 circularization, and urea-PAGE gel extraction were optimized.

Main Results:

  • The optimized method successfully produced precise linear precursor RNAs with correct ends.
  • The combination of GMP-primed transcription, T4 RNA ligase 2, and urea-PAGE yielded the highest fidelity circRNAs.
  • This workflow minimizes sequence and structural integrity issues in circRNA production.

Conclusions:

  • The developed methods enable the production of precise and homogenous engineered circRNAs.
  • These findings advance the potential of circRNAs as stable therapeutic agents.
  • The optimized protocol supports accurate functional studies using engineered circRNAs.