Triple helix conformation-specific blinking of Cy3 in DNA

Kawai, Kiyoshi; Maruyama, Atsushi

doi:10.1039/c5cc00607d

Cited by 15 publications

(19 citation statements)

References 39 publications

(53 reference statements)

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“…Previous investigations into the mechanism of fluorescence blinking of cyanine dyes used for super‐resolution imaging noted light‐induced isomerization as a side‐effect . Although photo‐isomerization serves the purpose of switching the fluorophore between bright and dark states, concerns have been raised about the timescale of such events. The photo‐isomers formed from cyanine dyes tend to revert back to the original structure on the timescale of a few hundred μs, depending on the molecular structure and local viscosity.…”

Section: Resultsmentioning

confidence: 99%

Photo‐isomerization of the Cyanine Dye Alexa‐Fluor 647 (AF‐647) in the Context of dSTORM Super‐Resolution Microscopy

Karlsson

Laude

Hall

et al. 2019

Chemistry A European J

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Cyanine dyes, as used in super‐resolution fluorescence microscopy, undergo light‐induced “blinking”, enabling localization of fluorophores with spatial resolution beyond the optical diffraction limit. Despite a plethora of studies, the molecular origins of this blinking are not well understood. Here, we examine the photophysical properties of a bio‐conjugate cyanine dye (AF‐647), used extensively in dSTORM imaging. In the absence of a potent sacrificial reductant, light‐induced electron transfer and intermediates formed via the metastable, triplet excited state are considered unlikely to play a significant role in the blinking events. Instead, it is found that, under conditions appropriate to dSTORM microscopy, AF‐647 undergoes reversible photo‐induced isomerization to at least two long‐lived dark species. These photo‐isomers are characterized spectroscopically and their interconversion probed by computational means. The first‐formed isomer is light sensitive and transforms to a longer‐lived species in modest yield that could be involved in dSTORM related blinking. Permanent photobleaching of AF‐647 occurs with very low quantum yield and is partially suppressed by the anaerobic redox buffer.

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Section: Resultsmentioning

confidence: 99%

Photo‐isomerization of the Cyanine Dye Alexa‐Fluor 647 (AF‐647) in the Context of dSTORM Super‐Resolution Microscopy

Karlsson

Laude

Hall

et al. 2019

Chemistry A European J

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…Blinking is an intrinsic characteristic of single-molecule emission and thus is often treated as evidence of observation of as ingle molecule. Various off-states that induceb linkingh ave been reported:1 )the triplets tate resulting from intersystem crossing, [32,33] 2) the reduced or oxidized state generated by photoinduced electron transfer, [34,35] 3) the isomerized state formed by trans-cis isomerization, [36,37] 4) the photoinduced thiol addition product typically seen in cyanine dyes, [38] 5) the formation of an on-fluorescent complex by association with aq uencher [39] and 6) quenching by an enzymatic reaction. [40] Blinking complicates interpretationo fs ingle-molecule measurements, and strategies to preventb linkingh ave been developed.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

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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“…KACB based on the formation of the following OFF states has been reported: a) at riplet state resulting from intersystem crossing, [30] b) a reduced state triggered by photoinduced electron transfer, [31][32][33] c) an isomerized state formed by trans-to-cis photoisomerization. [34] Since these OFF states are triggered by photoirradiation and hence the t ON value varies depending on the power intensity of the irradiation laser or the excitation efficiency,m icroenvironmental changes around the fluorophore are monitored by the t OFF .K ACBa llowed us to discriminate between DNAc onformations around af luoro-phore,s uch as hairpin loops,d uplexes,b ulged duplexes,a nd triplexes.H owever,t hese measurements were performed under ensemble conditions,a nd the KACB has not been proven to be applicable to single-molecule detection and analysis.H erein, we show that redox-blinking-based KACB (rKACB) can be utilized for real-time single-molecule monitoring of the structural switching dynamics of nucleic acids between ahairpin loop and as tem structure.…”

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confidence: 99%

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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Triple helix conformation-specific blinking of Cy3 in DNA

Cited by 15 publications

References 39 publications

Photo‐isomerization of the Cyanine Dye Alexa‐Fluor 647 (AF‐647) in the Context of dSTORM Super‐Resolution Microscopy

Photo‐isomerization of the Cyanine Dye Alexa‐Fluor 647 (AF‐647) in the Context of dSTORM Super‐Resolution Microscopy

Fluorescence Redox Blinking Adaptable to Structural Analysis of Nucleic Acids

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

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