Control of Triplet Blinking Using Cyclooctatetraene to Access the Dynamics of Biomolecules at the Single‐Molecule Level

Abstract

To explore the dynamics of biomolecules,tracing the kinetics of photo-induced chemical reactions via the triplet excited state (T 1 )o fp robe molecules offers at imescale that is about 10 6 times wider than via the singlet excited state (S 1 ). Using cyclooctatetraene (COT) as atriplet energy acceptor and at the same time as ap hotostabilizer,t he triplet-triplet energy transfer (TTET) kinetics governed by oligonucleotide (oligo) dynamics were studied at the single-molecule level by measuring fluorescence blinking. TTET kinetics measurement allowed us to access the length-and sequence-dependent dynamics of oligos and realize the single-molecule detection of am odel microRNAb iomarker.I ns harp contrast to the singlet-singlet Fçrster resonance energy transfer (FRET) that occurs in the 1-10 nm range,T TET requires aV an der Waals contact. The present method is thus ac omplementary method to FRET and provides direct information on biomolecular dynamics on the mst om st imescale.

Photochemical Mechanisms of Fluorophores Employed in Single‐Molecule Localization Microscopy

“…Nitrophenylalanine and Trolox have been used in a similar fashion to greatly enhance the photostability but are also not appropriate for photoswitching techniques [161] . Kawai and co‐workers used COT with ATTO 647N to control blinking for imaging the single‐molecule dynamics of biomolecules [162] . Increasing the photostability can enable the number of switching cycles to be increased.…”

Programmed Control of Fluorescence Blinking Patterns based on Electron Transfer in DNA

“…τ OFF is not affected by the laser dose, and thus it provides a robust basis for an analytical method. By controlling and observing fluorescence blinking triggered by photoisomerization, [15] triplet‐triplet energy transfer, [16,17] and inter‐ [18,19] and intra‐molecular [20,21] photoinduced electron transfer (ET) reactions, we were able to discriminate among DNA single‐stranded, double‐stranded, triple‐stranded, and hairpin loop structures, detect target DNA/RNA and point mutations, and measure DNA/RNA dynamics. Therefore, by creating fluorescent labels that blink at various values of τ OFF , it is possible to characterize multiple targets by analyzing the resulting blinking pattern.…”