Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

Abstract

Single‐molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around a fluorophore (rKACB), thereby enabling real‐time single‐molecule measurement of the structure‐changing dynamics of nucleic acids.

Fluorescence Redox Blinking Adaptable to Structural Analysis of Nucleic Acids

“…Blinking measurements : Fluorescence time traces and FCS were measured with a previously described confocal microscope system . Briefly, the system consists of an inverted optical microscope (IX73, Olympus) and 532 nm CW laser (OBIS 532 LS, Coherent).…”

“…Blinking measurements:F luorescence time traces and FCS were measured with ap reviously described confocal microscope system. [46,60] Briefly,t he system consists of an inverted optical microscope (IX73, Olympus) and 532 nm CW laser (OBIS 532 LS, Coherent). The output of the laser with 2mWo f5 32 nm laser light was focused by an objective (UPlanSApo 60X Water,N.A.…”

“…We recognized that the lifetime of the off‐state τ off in blinking depends on the environment and sought to take advantage of this to gain insight into the environment around a fluorescent molecule. We have developed a kinetic analysis based on the control of blinking (KACB) and have utilized KACB for structure analysis of nucleic acids . KACB requires a very small sample and can be used to determine structural switching rate and duration.…”

“…We have developed ak inetic analysis based on the control of blinking (KACB) and have utilized KACB for structure analysiso fn ucleic acids. [35,37,[43][44][45][46] KACB requires av ery smalls ample and can be used to determine structurals witching rate and duration. While FRET requires labelling of two fluorophores, KACB needs only as ingle fluorophore and is easy to implement.…”

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

“…[40][41][42][43][44] Normally this requires less than 1pmole of sample.B yi mmobilizing the molecule on ag lass surface, blinking from as ingle fluorescent molecule can be traced. [28] Thus T 1 -based kinetics addressed by fluorescence blinking measurement can be the basis of ultrasensitive analytic and diagnostic applications.…”