Real-Time Monitoring Of Higher-Order Structure Of RNAs By Temperature Dependent Chromatography And Spectroscopy

As RNA therapeutics continue to evolve, understanding the higher order structure (HOS) of complex oligonucleotides like single guide RNAs (sgRNAs) has become critical to optimizing their function and stability. However, many traditional analytical techniques fall short when it comes to resolving conformational and aggregation complexities in structurally complex RNAs.

In this session, we’ll explore a new biophysical characterization strategy that combines chromatography and microfluidic modulation spectroscopy (MMS) to investigate structural changes in two 100-mer sgRNAs. Using orthogonal methods including IP-RPLC, IEX, HILIC, SEC with MALS and MS, and isothermal MMS, our team evaluated aggregation species, conformational dynamics, and thermal transitions.

Key highlights include:

• Development of temperature-course SEC and thermal ramping MMS to track real-time HOS changes from ambient conditions to RNA melting points
• Discovery of two-step thermal dissociation: higher-order aggregate (HOA) dissociation followed by RNA dimer dissociation
• Thermodynamic profiling using SEC to calculate enthalpy and entropy changes
• Identification of infrared spectral markers in MMS linked to Watson–Crick base pairing and RNA HOS shifts

Join us to learn how this integrated toolkit can enhance RNA therapeutic candidate screening and formulation development through a deeper understanding of RNA structure under native conditions.