高田 忠雄

Protoporphyrin IX (PpIX) is a key endogenous photosensitizer used in photodynamic diagnosis (PDD) and photodynamic therapy (PDT); however, its poor aqueous solubility and strong aggregation markedly suppress fluorescence emission and singlet oxygen ( ¹ O ₂ ) generation. The mechanisms by which PpIX is solubilized intracellularly and becomes photodynamically active remain unclear. PpIX is predominantly produced in mitochondria, where mitochondrial DNA encodes numerous potential G‐quadruplex (GQ)‐forming sequences that can be transcribed into RNA. Although DNA GQs interact with porphyrins, the role of RNA GQs in intracellular PpIX solubilization remains largely unexplored. Here, we investigated the interaction of PpIX with DNA and RNA GQs and evaluated their ability to solubilize PpIX and restore its photodynamic activity using steady‐state and time‐resolved spectroscopic techniques. Incubation of aggregated PpIX with pre‐formed DNA or RNA GQs induced pronounced dispersion and solubilization, accompanied by the appearance of well‐defined Soret and Q bands and substantial fluorescence enhancement. Time‐resolved ¹ O ₂ luminescence measurements revealed efficient ¹ O ₂ generation from GQ‐bound PpIX, with apparent quantum yields of 0.17 and 0.10 for DNA‐GQ– and RNA‐GQ–bound PpIX, respectively. These results demonstrate that RNA GQs can function as effective endogenous solubilizing platforms for PpIX, providing new insights into the molecular basis of PpIX‐based PDD and PDT.

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.

B- to Z-DNA transitions play a crucial role in biological systems and have attracted the interest of researchers for their applications in DNA nanotechnology. DNA and DNA analogues have also been used as templates to construct helical chromophore associations with π interactions. In this work, the B- to Z-DNA transition-induced switching of pyrene in an association manner was evaluated using DNA duplexes with non-nucleosidic pyrene residues in the middle of d(CG) repeat sequences. One of the pyrene-labeled DNAs was shown to exhibit inverted exciton coupled circular dichroism signals upon pyrene association through a B- to Z-DNA transition. This observation indicates that pyrene association switches the DNA conformation from right- to left-handed. Interestingly, the fluorescence of the pyrene-labeled DNA duplex also dynamically changed upon switching of the pyrene in an association-based manner. Taken together, these studies demonstrate that pyrene-labeled DNA shows promise as a chiroptical molecular switch.

The construction of zipper-like chromophore-arrays in the major groove of duplex DNA remains a challenge because only a few chromophores for this application have been discovered. To address the challenge, dual-chromophore labeled DNAs having a self-complementary sequence were prepared using a solid-phase, post-synthetic, copper-catalyzed, alkyne-azide cycloaddition. The resulting chromophore-arrays on the labeled DNA duplexes were characterized. The dual-tetraphenylethene (TPE) or dual-pyrene (Py) labeled DNA formed self-complementary B-form duplexes and resulted in the construction of chromophore-arrays in the major groove. The TPE-arrays, in which TPEs were arranged in a zipper-like fashion, slightly destabilized the duplex because of their bulkiness and exhibited aggregation-induced-emission. The Py-arrays, in which Pys were not arranged in a zipper-like fashion, had no effect on duplex stability and exhibited weak excimer emission because Py was sufficiently small for free rotation in the major groove.

The construction of one-dimensional chromophore aggregates of naphthalenediimide (NDI) and bis(2-thienyl)diketopyrrolopyrrole (TDPP) using 40-mer oligodeoxythymidines (dT40) as a scaffold was previously reported. Furthermore, the chromophore-aggregate (NDI-dT40/TDPP-dT40) co-immobilized heterojunction gold electrode exhibited a more efficient photocurrent than the TDPP?dT40-immobilized electrode with selective photoexcitation of TDPP-dT40. In this work, a system comprising three components (in which the chromophore aggregates of diphenyl-diketopyrrolopyrrole (PDPP-dT40) are employed as a third component) was examined in order to enhance photocurrent efficiency. Selective photoexcitation of TDPP-dT40 on the NDI-dT40/TDPP-dT40/PDPP-dT40 electrode shows photocurrent responses with greater quantum yield than those of the TDPP-dT40, NDI-dT40/TDPP-dT40 and TDPP-dT40/PDPP-dT40 electrodes. The results suggest the addition of PDPP-dT40 can control charge separation and charge recombination between the electron and the hole (generated by an electron transfer from the photoexcited TDPP-dT40 to NDI-dT40).The construction of one-dimensional chromophore aggregates of naphthalenediimide (NDI) and bis(2-thienyl)diketopyrrolopyrrole (TDPP) using 40-mer oligodeoxythymidines (dT40) as a scaffold was previously reported. Furthermore, the chromophore-aggregate (NDI-dT40/TDPP-dT40) co-immobilized heterojunction gold electrode exhibited a more efficient photocurrent than the TDPP?dT40-immobilized electrode with selective photoexcitation of TDPP-dT40. In this work, a system comprising three components (in which the chromophore aggregates of diphenyl-diketopyrrolopyrrole (PDPP-dT40) are employed as a third component) was examined in order to enhance photocurrent efficiency. Selective photoexcitation of TDPP-dT40 on the NDI-dT40/TDPP-dT40/PDPP-dT40 electrode shows photocurrent responses with greater quantum yield than those of the TDPP-dT40, NDI-dT40/TDPP-dT40 and TDPP-dT40/PDPP-dT40 electrodes. The results suggest the addition of PDPP-dT40 can control charge separation and charge recombination between the electron and the hole (generated by an electron transfer from the photoexcited TDPP-dT40 to NDI-dT40).

We describe an approach to the development of a new electronic aptamer-based biosensor for multiprotein targets on a single platform. The multiple protein biosensing involves the co-adsorption of several thiol-terminated capture DNA sequences on a gold surface, along with the hybridization of redox-tagged aptamer to each corresponding capture sequences on the electrode, addition of target proteins, and monitoring aptamer-strand release through electrochemical measurement.