Scanning Single-Molecule Fluorescence Correlation Spectroscopy Enables Kinetics Study of DNA Hairpin Folding with a Time Window from Microseconds to Seconds

Bi, Hui-Min; Yin, Yandong; Pan, Bailong; Lu, Geng; Zhao, Xin

doi:10.1021/acs.jpclett.6b00720

Cited by 18 publications

(40 citation statements)

References 39 publications

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“…This indicates that the reaction rate constants do not depend strongly on the structure of the oligonucleotide and that the fluorescent molecules are not protected from solvent in the duplex (Figure b). This result is consistent with previous studies that indicated that rhodamine did not stack with the duplex . As k ss / k ds is slightly smaller than unity, the reaction of the fluorescent molecule and oxidant is faster in the double‐stranded than in the single stranded oligonucleotide.…”

Section: Resultssupporting

confidence: 92%

“…This result is consistent with previous studies that indicated that rhodamine did not stack with the duplex. [52] As k ss /k ds is slightly smaller than unity,t he reaction of the fluorescent molecule and oxidant is faster in the double-stranded than in the single stranded oligonucleotide. The fluctuating conformation of the single strand may slightly inhibit the oxidantf rom approachingt he fluorescent molecule.…”

Section: Discrimination Betweens Ingle-strandedand Double-stranded Stmentioning

confidence: 99%

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Fluorescence Redox Blinking Adaptable to Structural Analysis of Nucleic Acids

Miyata

Shimada

Maruyama

et al. 2018

Chemistry A European J

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The phenomenon of blinking is unique to single-molecule fluorescence measurements. By designing a fluorophore with an appropriate dark-state lifetime τ , a kinetic analysis based on the control of fluorescence blinking (KACB) was devised to investigate the dynamics of biomolecules. By controlling the redox-reaction-based blinking (rKACB), conformational dynamics of RNA at the single-molecule level was previously investigated. However, there is little knowledge about suitable fluorescent molecules for rKACB, and the application of rKACB has been limited to the analysis of hairpins and duplex structures of nucleic acids. In this work, various fluorescent molecules, including Alexa 488, R6G, TAMRA, ATTO 647N and ATTO 655, were evaluated for rKACB. Moreover, rKACB was adapted to the discrimination of DNA/DNA and DNA/RNA nucleic acid duplexes and investigation of antigen-antibody interactions. By changing the size of the oxidant, it was possible to determine the solvent accessibility of the target domain of the analyzed biomolecules.

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Section: Resultssupporting

confidence: 92%

Section: Discrimination Betweens Ingle-strandedand Double-stranded Stmentioning

confidence: 99%

Fluorescence Redox Blinking Adaptable to Structural Analysis of Nucleic Acids

Miyata

Shimada

Maruyama

et al. 2018

Chemistry A European J

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“…[31] This difference between R6G and ATTO 655 may stem from incomplete stacking of rhodamine dyes in the context of ad uplex structures,a ss uggested in previous studies. [33,46] This is in line with our previous report that the nature of the fluorophore used is critically important for sensitive read-out of the microenvironment around the fluorophore in rKACB. [31] To investigate whether rKACB is effective at monitoring the conformational switching dynamics of nucleic acids,w e performed real-time single-molecule analysis of conformational switching of the preQ 1 riboswitch from Fusobacterium nucleatum mRNA.…”

Section: Angewandte Chemiesupporting

confidence: 89%

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

et al. 2017

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Single-molecule fluorescence resonance energy transfer (smFRET) is ap owerful tool to investigate the dynamics of biomolecular events in real time.H owever,i t requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules.Herein, we introduce amethod for kinetic analysis based on control of fluorescence blinking (KACB), ag eneral approach to investigate the dynamics of biomolecules by using as ingle fluorophore.B yc ontrolling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around af luorophore (rKACB), therebye nabling real-time single-molecule measurement of the structure-changing dynamics of nucleic acids.Advances in single-molecule fluorescence microscopy in the last two decades have allowed us to reveal many important biochemical and cellular processes. [1][2][3][4][5] Single-molecule fluorescence measurement is especially effective for investigating the dynamic reaction and motion of biomolecules. [6][7][8][9][10] One of the most successful approaches is the observation of fluorescence resonance energy transfer between as ingle pair of fluorophores (smFRET). [11][12][13][14] Thed ynamics of distance changes between two sites on biomolecules or between two different molecules can be measured by monitoring timedependent fluctuation in the donor and acceptor fluorescence signals.T his information is usually inaccessible in ensemble measurements due to al ack of synchronization of dynamic motions or of the reactions of biomolecules.While smFRET is ap owerful tool and offers fruitful information on various dynamic reactions and the motions of biomolecules,itnevertheless has two disadvantages.O ne is that it can be applied only to dynamics that accompany large distance changes between two fluorophores (> 2nm). Theother is the need to label biomolecules with two fluorophores.T hus the development of am ethod that requires the labeling of only as ingle fluorophore to provide dynamic information on biomolecules is highly desired.To date,c hemists have developed various environmentsensitive fluorophores. [15][16][17][18][19][20] These fluorophores undergo fluorescence intensity and lifetime changes or spectral shifts related to microenvironmental changes around the fluorophore.E nvironment-sensitive changes in the fluorescence intensity and lifetime of Cy3 have been successfully applied to the observation of the dynamics of protein-nucleic acid interactions. [21,22] However, it is still often challenging to achieve sufficient time resolution in single-molecule dynamic analysis based on environment-sensitive fluorophores.Fluorescent signals from asingle fluorophore often blink, reflecting time-dependent fluctuations between bright (ON) and dark (OFF) states. [23][24][25][26] While background fluorescence is ac ommon obstacle in single-molecule fluorescence microscopy,asingle fluorescence molecule exhibiting ac ontrolled unique blinking pattern can be readily read ...

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“…To nd out whether the single FRET peak represents a single half-open state or is due to the fast interchange between the open and closed states, we conducted the FRET-FCS experiment on a home-built scanning confocal microscope. 16 No dynamic relaxation process was observed in 90-210_F-DNA Me (Fig. S10 †), conrming that the FRET peak signals a single half-open state.…”

mentioning

confidence: 99%