Real-Time Monitoringof Higher-Order Structure ofRNAs by Temperature-Course Size Exclusion Chromatography and MicrofluidicModulation Spectroscopy

Recently, there has been emerging interest in the characterization of the higher order structure (HOS) of oligonucleotide therapeutics because of its potential impact on the function. However, many existing experimental and computational methods face challenges with respect to throughput, cost, and resolution for large ribonucleic acids (RNAs). In this study, we present the use of two orthogonal analytical methods, size-exclusion chromatography (SEC) and microfluidic modulation spectroscopy (MMS), which are used to investigate conformational changes of two 100 mer single guide RNAs (sgRNAs) with complex HOS and aggregation specie profiles. SEC, coupled with multiangle light scattering (MALS), mass spectrometry (MS), and isothermal MMS revealed various forms of aggregation and potential interactions. We also developed temperature-course SEC and thermal ramping MMS methods to monitor real-time HOS changes from room temperature to the RNA melting point. Through the experiments, we observed two discrete steps of thermally induced dissociation of RNA aggregates, namely higher order aggregates (HOA) dissociation and dimer dissociation. Temperature-course SEC allows for thermodynamic analysis of the enthalpy and entropy of the reaction. We also identified two spectral regions in infrared (IR) spectra with thermal ramping MMS, 1665 cm^–1 and between 1700 and 1720 cm^–1, which closely correlated to the Watson–Crick base pairing and the related HOS change in RNA. The combination of SEC and MMS offers a comprehensive biophysical characterization toolkit for RNA HOS under native conditions, providing valuable insights for candidate optimization and formulation screening in the development of RNA therapeutics.