Second-harmonic generation-based methods to detect and characterize ligand-induced RNA conformational changes

被引：6

作者：

Birman, Yuliya ^{[1
]}

Khorsand, Sina ^{[1
]}

Tu, Erick ^{[1
]}

Mortensen, Richard B. ^{[1
]}

Butko, Margaret T. ^{[1
]}

机构：

[1] Biodesy Inc, 170 Harbor Way Suite 100, San Francisco, CA 94080 USA

来源：

METHODS | 2019年 / 167卷

关键词：

SMALL MOLECULES; BINDING; INHIBITORS; SHG;

D O I：

10.1016/j.ymeth.2019.05.012

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Second-harmonic generation (SHG) is a biophysical tool that senses ligand-induced conformational changes in biomolecules. The Biodesy Delta (TM) 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.

引用

页码：92 / 104

页数：13

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