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Probing the charge-transfer dynamics in DNA at the single-molecule level.

Journal of the American Chemical Society (2011-08-31)
Kiyohiko Kawai, Eri Matsutani, Atsushi Maruyama, Tetsuro Majima
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 655, BioReagent, suitable for fluorescence, ≥85% (HPLC)
Sigma-Aldrich
Atto 655 NHS ester, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 655-Biotin, BioReagent, suitable for fluorescence, ≥95.0% (HPCE)
Sigma-Aldrich
Atto 655 amine, suitable for fluorescence
Sigma-Aldrich
Atto Oxa12 NHS ester
Sigma-Aldrich
Atto Oxa12 Biotin
Sigma-Aldrich
Atto 655-maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling rate)

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