高田 忠雄

DNA charge transfer highly depends on the electronic interaction between base pairs and reflects the difference in the base position and sequence. For the purpose of investigating the transfer process of individual DNA molecules and the optical readout of DNA information at the single-molecule level, performed single-molecule observation of the DNA charge process by using single-molecule fluorescence spectroscopy. DNA charge transfer process, leading to the oxidation of fluorescent dye, was explored by monitoring the on-off signal the dye after the charge injection by the excitation of a sitizer. The photobleaching efficiency of the dyes by the charge transfer specifically depended on the base sequence and mismatch base pair, demonstrating the discrimination of the individual DNA information. Based on this approach, the optical readout of a single-base mismatch contained in a target DNA was performed at the single-molecule level.

(Figure Presented) Got a light? The importance of the relationship between the charge recombination and charge transfer during photocurrent generation through DNA films is described. The photocurrent efficiency for DNA films, in which the charge-transfer and recombination rates were modulated by changing the sequence, was investigated by using the photoelectrochemical measurements on an Au electrode. © 2007 Wiley-VCH Verlag GmbH &amp Co. KGaA.

A series of naphthalimicle (Nl)- and 5-bromocytosine (C-br)-modified oligodeoxynucleotides (ODNs) were prepared, and their lifetimes of the charge-separated states during the photosensitized one-electron oxidation of DNA were measured. Various lifetimes of the charge-separated states were observed depending on the sequence and the incorporation sites of C-br, and the oxidation potential of G in the C-br:G base-pair relative to that of G in the C:G base-pair and in the GGG sequence was determined by comparing the lifetimes of the charge-separated states. The change in the cytosine C5 hydrogen to bromine resulted in a 24 mV increase in the oxidation potential of G in the C-br:G base-pair as compared to that of G in the C:G base-pair, the value of which is comparable to a 58 mV decrease in the oxidation potential of G in the GGG sequence. These results clearly demonstrate that hole transfer in DNA can be controlled through hydrogen bonding by introducing a substituent on the cytosine.

Doubly internally pyrene (Py) modified oligodeoxynucieotides (ODNs) were synthesized, and the formation rates of the Py dimer radical cation (PY2.+) were measured upon one-electron oxidation during pulse radiolysis. The formation Of Py-2(.+) with an optical absorption at 1550 nm (charge resonance band) was observed in the time range of 1 mus to 1 ms after an electron pulse during the pulse radiolysis of a D2O solution of the doubly internally Py modified ODN in the presence of K2S2O8. The formation rate Of Py-2(.+) in DNA reflected the dynamics of the DNA, which allows the interaction between Py.+ and Py, and was affected by the distance between the two Py's and the local environment of each Py. The trapping of the transiently formed DNA conformation by the attractive charge resonance interaction was demonstrated to be useful to obtain structural and dynamic information of the fluctuating DNA in the time range of 1 mus to 1 ms.

Mechanism of photo-induced electron transfer and the subsequent hole transfer in DNA has been studied extensively, but so far we are not aware of any reliable report of the observation of the long-distance hole-transfer process. In this article, we demonstrate the results of direct observation for the long-distance hole transfer in double-helical DNA over 100 Angstrom with time-resolved transient absorption measurements. DNA conjugated with naphthalimide (NI) and phenothiazine (PTZ) (which worked as electron-acceptor and donor molecules, respectively) at both 5'ends was synthesized to observe the hole-transfer process. Site-selective charge injection into G by means of the adenine-hopping process was accomplished by excitation of NI with a 355-nm laser flash. Transient absorption around 400 nm, which was assigned to the NI radical anion, was observed immediately after the irradiation of a laser flash, indicating that the charge separation between NI and the nearest G occurred. Then, the transient absorption of the PTZ radical cation (PTZ(.+)) at 520 nm was emerged, which was attributed to the hole transfer through DNA to the PTZ site. By monitoring the time profiles of the transient absorption of PTZ(.+) for NI-A(6)-(GA)(n)-PTZ and NI-A(6)-(GT)(n)-PTZ (n = 2, 3, 4, 6, 8, 12) (base sequences correspond to those for DNA modified with NI), the long-distance hole-transfer process from G to PTZ, which occurred in the time scale of microsecond to millisecond, was observed directly. By assuming an average distance of 3.4 Angstrom between base-pairs, total distance reaches 100 Angstrom for n = 12 sequences. Our results clearly show the direct observation of the long-distance hole transfer over 100 Angstrom.

Charge transfer in DNA is of current interest because of the involvement of charge transfer in oxidative DNA damage and electronic molecular devices. We have investigated the charge separation process via the consecutive adenine (A)-hopping mechanism using laser flash photolysis of DNA conjugated with naphthaldiimide (NDI) as an electron acceptor and phenothiazine (PTZ) as a donor. Upon the 355-nm laser flash excitation of NDI, the charge separation and recombination process between NDI and PTZ was observed. The yields of the charge separation via the consecutive A-hopping were slightly dependent upon the number of A bases between the two chromophores, while the charge recombination rate was strongly dependent upon the distance. The charge-separated state persisted over 300 mus when NDI was separated from PTZ by eight A bases. Furthermore, the rate constant of the A-hopping process was determined to be 2 x 10(10) s(-1) from an analysis of the yield of the charge separation depending on the number of A-hopping steps.

Three naphthalimide (NI) derivatives (NIN, NIP, and NIO) as photosensitizers for the G-selective oxidation were synthesized, and the mechanism of the photosensitized one-electron oxidation of DNA by the NI derivatives was studied. NIN possessing a cationic side chain exhibited strong association with the olizodeoxynucleotides (ODNs) due to an electrostatic interaction between the phosphate groups of ODN and the cationic group of NIN, while no association was observed for NIP possessing an anionic side chain due to electrostatic repulsion among the phosphate groups. The DNA-binding properties of NIO with a neutral side chain were intermediate between those of NIN and NIP. The effects of the electrostatic interaction and repulsion between the NI derivatives and ODN and stacked G bases on the photosensitized one-electron oxidation of ODN were studied by the nanosecond transient absorption measurement and HPLC analysis. The yield of the NI derivatives in the triplet excited state ((NI)-N-3*) observed immediately after a 355-nm laser flash decreased with an increase in the binding constants, and almost no transient absorption was observed when NIN was used as a photosensitizer. This result indicates that rapid charge separation occurs between the NI in the singlet excited state ((1)Nl*) and the adjacent nucleobase, followed by a rapid charge recombination between the NI radical anion and the adjacent nucleobase radical cation when NIN is bound to ODN. Therefore, the sequence dependence of the one-electron oxidation of ODN was investigated using NIP, which does not bind to ODN. By monitoring the one-electron reduced form of NIP (NIP.-) produced by the electron transfer from ODN to (NIP)-N-3*, we found that the charge-separation efficiency increased with the sequence of sequential G's, such as GG and GGG, because of the oxidation potential of G decreased by the stacking effect of G's. To clarify the relationship between the formation of the transient intermediates observed during the LFP and actual amount of ODN oxidative damage, we performed the quantitative analysis of the ODN oxidative lesion caused by the NI derivatives using HPLC. With respect to the sequence of ODN, larger amounts of G consumed by the photosensitized one-electron oxidation were observed with ODN possessing multiple G, GG, and GGG compared to a single G. In contrast to the LFP experiments, a similar amount of oxidative damage was observed for three synthetic NI derivatives, indicating that the association of the NI derivatives to ODN apparently has no effect on the one-electron oxidative process. This might be due to the O-2 involved in the one-electron oxidation process. These results are discussed in the context of the concentration of 02 in the solution.

To clarify the hole-transfer mechanism in DNA and the factor controlling the hole-transfer rates, the kinetics of hole transfer in DNA was studied by monitoring the transient absorption of the pyrene (Py) radical cation (Py.+) during the pulse radiolysis of oligodeoxynucleotides (ODNs) conjugated with Py. By analyzing the transient absorption of Py.+ formed by the hole transfer from DNA to Py, the rate constants of the hole transfer in various sequences of DNA were determined. The rate constants of the hole transfer from the nearest guanine (G) to Py were weakly dependent upon the distance between Py and the nearest G, indicating the occurrence of the multistep hole transfer in DNA. In contrast, in the hole transfer where the rate-determining step was the single-step hole transfer between Gs, the rate was strongly dependent upon the distance between the Gs. Comparing the intervening nucleobases between Gs, the rate constants of the multistep hole transfer increased in the order G(.+)AG > G(.+)AC > G(.+)TG. These results showed that the hole transfer between Gs was effectively mediated when the bridge was A. The effect of multiple Gs (GGG) on multistep hole transfer was examined. The hole transfer from DNA to Py was slowed by the presence of the GGG site that was far away from Py, indicating that the multiple G worked as a hole trap site. Our results are discussed in the context of the previously reported theoretical and experimental results.

Hole transfer process in ODNs conjugated with two organic molecules, pyrene (Py) and phenothiazine (Ptz) was investigated with the pulse radiolysis measurements. Monitoring the transient absorption of Py.+ and Ptz(.+), it was shown that the hole transfer rate was dependent on the distance and sequence between Py and Ptz. (C) 2003 Elsevier Science Ltd. All rights reserved.