Probing the Charge-Transfer Dynamics in DNA at the Single-Molecule Level

Abstract

Photoinduced charge-transfer fluorescence quenching of a fluorescent dye produces the nonemissive charge-separated state, and subsequent charge recombination makes the reaction reversible. While the information available from the photoinduced charge-transfer process provides the basis for monitoring the microenvironment around the fluorescent dyes and such monitoring is particularly important in live-cell imaging and DNA diagnosis, the information obtainable from the charge recombination process is usually overlooked. When looking at fluorescence emitted from each single fluorescent dye, photoinduced charge-transfer, charge-migration, and charge recombination cause a "blinking" of the fluorescence, in which the charge-recombination rate or the lifetime of the charge-separated state (τ) is supposed to be reflected in the duration of the off time during the single-molecule-level fluorescence measurement. Herein, based on our recently developed method for the direct observation of charge migration in DNA, we utilized DNA as a platform for spectroscopic investigations of charge-recombination dynamics for several fluorescent dyes: TAMRA, ATTO 655, and Alexa 532, which are used in single-molecule fluorescence measurements. Charge recombination dynamics were observed by transient absorption measurements, demonstrating that these fluorescent dyes can be used to monitor the charge-separation and charge-recombination events. Fluorescence correlation spectroscopy (FCS) of ATTO 655 modified DNA allowed the successful measurement of the charge-recombination dynamics in DNA at the single-molecule level. Utilizing the injected charge just like a pulse of sound, such as a "ping" in active sonar systems, information about the DNA sequence surrounding the fluorescent dye was read out by measuring the time it takes for the charge to return.

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

“…[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.…”

“…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- …”

“…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.…”

“…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.…”

Photoinduced Charge-Separation in DNA

“…In this case A-T base-pairs serve as a redox inactive spacer that prevents the formation of a contact ion pair. A long-lived chargeseparated state was formed in DNA modified with a commonly used fluorophore such as TAMRA, Alexa 532, and ATTO 655, suggesting that other fluorescent dyes of which fluorescence is quenched only by G may also produce a long-lived charge-separated state in DNA upon the photoirradiation [25,26].…”