Abstract
RNA G-quadruplexes (G4s) play important roles in translational regulation, mRNA processing events and gene expression. Therefore, a fluorescent probe that is capable of efficiently recognizing RNA G-quadruplex structures among other RNA forms is highly desirable. In this study, a water-soluble fluorogenic dye (i.e., Thioflavin T (ThT)) was employed to recognize RNA G-quadruplex structures using UV–Vis absorption spectra, fluorescence spectra and emission lifetime experiments. By stacking on the G-tetrad, the ThT probe exhibited highly specific recognition of RNA G-quadruplex structures with striking fluorescence enhancement compared with other RNA forms. The specific binding demonstrates that ThT is an efficient fluorescence sensor that can distinguish G4 and non-G4 RNA structures.
Highlights
One of the most important types of nucleic acid structure is the G-quadruplex (G4), which is formed from four guanine bases by stacking of Hoogsteen bonded G-quartets[1]
Circular dichroism (CD) has been extensively used to monitor G-quadruplex formation because the positive band at 264 nm and the negative band at 240 nm indicate the formation of parallel-type G-quadruplex structures[16]
To investigate the feasibility of the ThT probe for RNA G-quadruplex structure recognition, we examined its selectivity towards various RNA forms including RNA G-quadruplex sequences (ADAM10, BCL-2, ERSI, TRF2, VEGF, C9orf[72] and ZIC1) and other RNA forms, such as transfer RNA, mutation sequences (ERSI-mut, C9orf72-mut), single-stranded, hairpin (HP18), double-stranded, threeway junction (3-WJ) and four-way junction (4-WJ)
Summary
One of the most important types of nucleic acid structure is the G-quadruplex (G4), which is formed from four guanine bases by stacking of Hoogsteen bonded G-quartets[1]. Many techniques including X-ray crystallography and NMR experiments have been utilized to identify high-resolution structures of RNA G-quadruplexes[14,15]. These techniques are more suitable for comprehensively studying targeted RNA G-quadruplex structures. Thioflavin T (ThT, Fig. 1), which is an extrinsic fluorescent probe for the identification of amyloid fibrils in previous studies[18,19], was selected to target RNA G-quadruplex using a high-throughput screening approach. The RNA G-quadruplex structure-selective binding of ThT was employed to recognize RNA G-quadruplex structures from other RNA forms via its fluorescence light-up (Fig. 2). The fluorescence intensity enhancement, which results from ThT binding to RNA G-quadruplex, is expected to provide another RNA G-quadruplex probe
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