Thioflavin T Is a More General and Less Interfering Probe for Aptamer Binding Assays Than SYBR Green I

作者：Yun Shu, Shiyuan Liu, Juewen Liu · 发表于：Analytical Chemistry · 年份：2025 · DOI：10.1021/acs.analchem.5c03351 · 被引用次数：33 · 研究领域：Advanced biosensing and bioanalysis techniques、DNA and Nucleic Acid Chemistry、RNA Interference and Gene Delivery

Label-free fluorescent binding assays employing DNA staining dyes as probes are widely adopted techniques in the aptamer field. While many dyes have been used, thioflavin T (ThT) did not receive much attention for this purpose until recently, since it has long been perceived primarily as a G-quadruplex staining dye. Based on recent studies, ThT appears to serve as a reliable probe for evaluating the binding of non-G-quadruplex aptamers, and we seek to clarify the underlying mechanisms responsible for the exceptional performance of ThT. We investigated three non-G-quadruplex aptamers targeting adenosine, kanamycin, and Tris(hydroxymethyl)aminomethane (Tris). Consistent K d values and fluorescence reductions were observed in the presence of ThT regardless of the aptamer-to-dye ratio. In contrast, SYBR Green I (SGI) exhibited responses with apparent K d values that were far away from the true K d in most cases. Additional assays demonstrated that ThT exhibits a weaker affinity for aptamers and random DNA sequences compared to SGI. In addition, ThT has weaker affinities to double-stranded DNA than to single-stranded DNA, which is opposite to that for SGI. Lastly, testing the T30695 DNA binding with Pb 2+ confirmed that ThT can influence the binding of G-quadruplex aptamers. These findings explain ThT’s reliability for probing non-G-quadruplex aptamers.