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

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Фев. 27, 2025

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

Язык: Английский

Identification of antibody-resistant SARS-CoV-2 mutants via N4-Hydroxycytidine mutagenesis

Antiviral Research, Год журнала: 2024, Номер 231, С. 106006 - 106006

Опубликована: Сен. 16, 2024

Язык: Английский