Tadao Takada

We investigate the transition processes between the emitting (ON) and non-emitting (OFF) states of fluorescent molecules using a machine-learning approach. In fluorescently labeled DNA, continuous fluorescence is observed under irradiation; however, the system occasionally transitions to a non-emitting state, often associated with a charge-separated configuration. The resulting fluorescence trajectories exhibit characteristic blinking behavior —alternating ON and OFF states— which is heavily obscured by various sources of noise, making reliable state classification challenging. Because such trajectories represent typical stochastic time-series data, advanced analytical techniques are required. In this study, we apply a hidden Markov model to extract hidden ON/OFF states from noisy fluorescence trajectories using the forward-filtered backward-blocking Gibbs sampling algorithm, and construct probability density functions of the ON- and OFF-state durations to characterize the blinking dynamics. From these distributions, the characteristic relaxation times are evaluated as 17.6 ms for the ON state and 7.8 ms for the OFF state. The relatively long OFF period indicates that the charge-separated state in the DNA-ATTO655 system is fairly stable, suggesting suppressed charge recombination. In addition, we discuss the characteristic timescale of the light absorption–emission process in the ON state in terms of the average photon count per time bin. These results provide new insights into the fluorescence dynamics of single DNA-fluorophore systems. Finally, we discuss the detailed conditions required for reliable time-series analysis in terms of the photon-count histogram shape and the time-bin width used in the trajectories.

Artificial DNA molecules functionalized with fluorescent dyes are useful for developing fluorescent chemosensors and molecular imaging tools. Fluorescent nucleic acids have been developed based on the excellent fluorescent properties of perylenediimide (PDI); however, the electron transfer quenching of PDI by purine bases limits the sequence design of fluorescent probes. In this study, to compensate for the disadvantages of PDI due to electron transfer quenching, we used PDI with a methoxy group introduced into the perylene ring (PO), synthesized PO-substituted nucleic acids, and investigated their fluorescence properties. The results showed that PO in the DNA exhibited longer-wavelength fluorescence than unsubstituted PDI and displayed strong fluorescence when surrounded by AT base pairs. We also found that PO exhibits a fluorescence on-off response to the hybridization reaction and selective quenching by guanine bases in the vicinity of PO, which could be applied to fluorescent biosensors for single nucleotide identification and nucleic acid detection. Because of the high photostability of PO, single-molecule measurements were feasible, allowing confirmation of PO's fluorescence switching behavior of PO in response to DNA conformational changes and the presence of guanine bases at the single-molecule level using fluorescence correlation spectroscopy (FCS).

Abstract Fluorescence imaging uses changes in the fluorescence intensity and emission wavelength to analyze multiple targets simultaneously. To increase the number of targets that can be identified simultaneously, fluorescence blinking can be used as an additional parameter. To understand and eventually control blinking, we used DNA as a platform to elucidate the processes of electron transfer (ET) leading to blinking, down to the rate constants. With a fixed ET distance, various blinking patterns were observed depending on the DNA sequence between the donor and acceptor units of the DNA platform. The blinking pattern was successfully described with a combination of ET rate constants. Therefore, molecules with various blinking patterns can be developed by tuning ET. It is expected that the number of targets that can be analyzed simultaneously will increase by the power of the number of blinking patterns.