Binding activated single molecule burst analysis highlights amyloid sensing interaction of dye SYPRO orange.

Mode of interaction between fluorescent probe and local binding site on fibril architecture is central to the design of efficient amyloid sensors. SYPRO orange (SO) is reported to have two binding modes with two distinct binding configurations, namely weakly bound inclined and strongly bound surface hugging populations. Though two distinct sites for non-radiative relaxation, i.e. central π-bridge site and the electron donor amino site is evident from the molecular framework, it is unclear which site dictates fluorescence enhancement upon binding. We recommend employing the inherent sensitivity of fluorescence lifetime to alterations in non-radiative pathways for different binding configurations. In this contribution an attempt has been made to segregate binding activated single molecule bursts recorded with pulsed excitation into requisite weak and strong binding events, which were subsequently correlated with their corresponding excited state lifetimes. Comparison of fluorescence decays for these two binding modes suggest minor role of non-radiative relaxation at π-bridge site in deciding excited state decay of bound SO molecules. On the contrary, plausible hydrophobic interaction of aliphatic tails at the electron donor site with the fibril imparts configurational restrain at the amino group site, hindering its non-radiative pathways leading to increased fluorescence intensity and lifetime. This sensing behaviour of SO is consistent for fibrils of amyloidogenic proteins lysozyme and insulin. Present work has the relevance in the rational design of amyloid sensor as well as better super-resolution imaging probes for localization microscopy.