Detection of Single‐Nucleotide Variations by Monitoring the Blinking of Fluorescence Induced by Charge Transfer in DNA

Kawai, Kiyoshi; Majima, Tetsuro; Maruyama, Atsushi

doi:10.1002/cbic.201300380

“…In KACB,t he nature of the OFF state plays the key role.T he OFF state should form reversibly and should persist longer than one microsecond. 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- …”

mentioning

confidence: 99%

“…In KACB,t he nature of the OFF state plays the key role.T he OFF state should form reversibly and should persist longer than one microsecond. 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.…”

mentioning

confidence: 99%

“…In KACB,t he nature of the OFF state plays the key role.T he OFF state should form reversibly and should persist longer than one microsecond. 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%

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

Kawai

¹

,

Miyata

²

,

Shimada

³

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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“…[1][2][3] During the repetitive cycles of excitation of the S 0 ground state to the singlet excited state (S 1 ) and subsequent emissions that allow a return to S 0 (on-state), fluorescent molecules occasionally become non-fluorescent (off-state). Among such phenomena, in our recent studies we have focused on the fluctuating emissions between bright ''on'' and dark ''off'' states of fluorescent molecules, or so-called ''blinking''.…”

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confidence: 99%

“…Potential transient dark off-states that may cause such blinking are (i) the triplet state resulting from the intersystem crossing, (ii) the reduced or the oxidized state triggered by electron transfer, (iii) formation of a non-fluorescent complex between a fluorescent molecule and a quencher, (iv) intramolecular spirocyclization, and (v) the isomerized state formed by photo-triggered trans-cis isomerization. 2 Recently, we reported that blinking triggered by the formation of the triplet state of R6G provides information regarding the solvent accessibility of the fluorescent molecule. [4][5][6] Based on the synthesis and observation of DNA site-specifically modified with a fluorescent molecule, we have shown that the charge-separation, charge-transfer, and charge-recombination dynamics in DNA can be measured by monitoring the blinking.…”

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confidence: 99%