Second-harmonic generation-based methods to detect and characterize ligand-induced RNA conformational changes
| Autor: | Yuliya Birman, Sina Khorsand, Erick Tu, Margaret T. Butko, Richard B. Mortensen |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
animal structures
Aptamer Ligands General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Theophylline Caffeine Drug Discovery Humans Molecular Biology 030304 developmental biology chemistry.chemical_classification 0303 health sciences Oligonucleotide Drug discovery Biomolecule 030302 biochemistry & molecular biology RNA Second-harmonic generation Ligand (biochemistry) High-Throughput Screening Assays chemistry Biophysics Nucleic Acid Conformation Conjugate |
| Zdroj: | Methods. 167:92-104 |
| ISSN: | 1046-2023 |
| Popis: | Second-harmonic generation (SHG) is a biophysical tool that senses ligand-induced conformational changes in biomolecules. The Biodesy Delta™ has been developed as a high-throughput screening platform to monitor conformational changes in proteins and oligonucleotides by SHG to support drug discovery efforts. This work will outline (1) an overview of this technology, (2) detailed protocols for optimizing screening-ready SHG assays on RNA targets, (3) practical considerations for developing robust and informative SHG measurements, and (4) a case study that demonstrates the application of these recommendations on an RNA target. The previously published theophylline aptamer SHG assay [1] was further optimized to maximize the assay window between the positive control (theophylline) and the negative control (caffeine). Optimization of this assay provides practical considerations for building a robust SHG assay on an RNA target, including testing for specific tethering of the conjugate to the surface as well as testing tool compound response stability, reversibility, and concentration-dependence/affinity. A more robust, better-performing theophylline aptamer SHG assay was achieved that would be more appropriate for conducting a screen. |
| Databáze: | OpenAIRE |
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