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Real-Time Monitoring of Higher-Order Structure of RNAs by Temperature-Course Size Exclusion Chromatography and

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Characterizing higher order structure (HOS) of RNA therapeutics is crucial. Size-exclusion chromatography (SEC) and microfluidic modulation spectroscopy (MMS) effectively analyze RNA aggregation and conformational changes.

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Area of Science:

  • Biophysical characterization
  • RNA therapeutics development
  • Analytical chemistry

Background:

  • Emerging interest in higher order structure (HOS) of oligonucleotide therapeutics due to functional impact.
  • Existing methods for characterizing large ribonucleic acids (RNAs) face throughput, cost, and resolution limitations.

Purpose of the Study:

  • To investigate conformational changes and aggregation of single guide RNAs (sgRNAs) using orthogonal analytical methods.
  • To develop and apply size-exclusion chromatography (SEC) and microfluidic modulation spectroscopy (MMS) for RNA HOS analysis.

Main Methods:

  • Utilized SEC coupled with multiangle light scattering (MALS) and mass spectrometry (MS).
  • Employed isothermal and temperature-course SEC, alongside thermal ramping microfluidic modulation spectroscopy (MMS).
  • Monitored real-time HOS changes from room temperature to RNA melting point.

Main Results:

  • Identified various forms of aggregation and potential interactions in 100 mer sgRNAs.
  • Observed two discrete steps of thermally induced dissociation: higher order aggregates (HOA) and dimer dissociation.
  • Correlated specific infrared spectral regions (1665 cm⁻¹, 1700-1720 cm⁻¹) with Watson-Crick base pairing and HOS changes.

Conclusions:

  • The combination of SEC and MMS provides a comprehensive toolkit for RNA HOS characterization under native conditions.
  • This approach offers valuable insights for optimizing RNA therapeutic candidates and screening formulations.
  • Demonstrated the utility of SEC and MMS for analyzing complex HOS and aggregation profiles in large RNAs.