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

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 <i>K</i>_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 <i>K</i>_d values that were far away from the true <i>K</i>_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 Pb²⁺ confirmed that ThT can influence the binding of G-quadruplex aptamers. These findings explain ThT's reliability for probing non-G-quadruplex aptamers.