中村 光伸

The molecular arrangement of functional chromophores is essential to construct functional nanomaterials. Synthetic DNA has been used as a structural scaffold to control and construct molecular assembly. We now describe the formation of artificial DNA/porphyrin complexes where porphyrins working as photoactive molecules were placed at specific locations on the DNA through a non-covalent interaction. Spectroscopic analysis by UV/vis, circular dichroism (CD), and melting temperature measurements showed that the binding of water-soluble porphyrin derivatives with methylpyridinium groups (TMPyP and DMPyP) to DNA can be directed by hydrophobic cavities composed of a pair of abasic site analogs (dS). CD measurements showed that the two porphyrins can be accommodated at two specific sites when the DNA molecule had two cavities, as evidenced by the exciton-coupled CD spectra of the porphyrins. Photoelectrochemical experiments showed that the DNA complexes accommodating DMPyP at specific locations can respond to light excitation to generate a photocurrent when immobilized on the electrode surface. Our results demonstrated that the control of the porphyrin binding using the dS/dS pair is a useful approach to construct artificial DNA with porphyrin molecules, leading to the design of photoresponsive materials and photoelectrochemical sensors.

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.

We here describe a photocurrent generation system exploiting gold nanoparticles (AuNPs) that cover perylenediimide-DNA complexes on electrode surfaces. Enhanced photocurrents were generated by the irradiation of the AuNPs, attributed to the efficient excitation of the perylenediimides by a local electric field on the surface of the AuNPs.

The preparation of homo- and heterocomplexes composed of the parent perylene diimide (PH) and pyrrolidine-substituted perylene diimide (PN) in DNA and rapid electron transfer in these complexes, which has been analyzed by steady-state fluorescence and femtosecond transient absorption measurements, have been demonstrated. The DNA molecules possessing PH and PN were prepared through a recently developed method that involved the reaction of enzymatically generated abasic sites with the amino groups of the perylene diimide molecules, through which these molecules can be incorporated as base surrogates within the DNA base stack. Melting temperature analysis showed that the PH and PN monomers, and their homo- and heterodimers, contribute to the stabilization of the DNA duplex, and is comparable to that of natural base pairs. Fluorescence measurements showed that PH in a single-stranded oligopyrimidine showed a strong fluorescence, whereas the fluorescence of PH was completely quenched upon pairing with PN (PH/PN) through duplex formation. The transient absorption measurements showed that a rapid electron-transfer reaction in the stacked PH/PN heterodimer occurred on a sub-picosecond timescale, which allowed highly efficient fluorescence quenching. The PH/PN pair, which served as a fluorophore and quencher, were utilized to design molecular beacon probes with a high signal/noise ratio. The PH/PN pair was also capable of forming a stable, stacked dimer structure and induced rapid electron transfer that could serve as a good signal reporter for fluorescent nucleic acid detection.

Abstract In this report, we describe the circularly polarized luminescence (CPL) of the RNA duplexes having one to four 2′‐O‐pyrene modified uridines (Upy) and the DNA duplexes having two, four, and six pyrene modified non‐nucleosidic linkers (Py). Both the pyrene π‐stack arrays formed on the RNA and DNA double helical structures exhibited pyrene excimer fluorescence. In the pyrene‐modified RNA systems, the RNA duplex having four Upys gives CPL emission with glum value of <0.01 at 480?nm. The structure of pyrene stacks on the RNA duplex may be rigidly regulated with increase in the Upy domains, which resulted in the CPL emission. In the DNA systems, the pyrene‐modified duplexes containing two and four Pys exhibited CPL emission with glum values of <0.001 at 505?nm. The pyrene π‐stack arrays presented here show CPL emission. However, the glum values are relatively small when compared with our previous system consisting of the pyrene‐zipper arrays on RNA.

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).

This work describes the formation of a porphyrin (Por) dimer using a DNA duplex as a scaffold and photocurrent generation from electrodes modified with a monolayer of Por–DNA conjugates. The solid-phase click reaction between an azide-porphyrin and oligonucleotide labeled with an ethynyl group on CPG support was utilized to conjugate the Por to the DNA. UV/Vis absorption and circular dichroism (CD) spectral studies revealed that the Por dimer can be formed through DNA hybridization and that through-space electronic interactions, characterized from the exciton-coupled absorption and the bisignate CD, can occur between the two Por molecules. Photoelectrochemical experiments were performed for the electrodes functionalized with a monolayer composed of the Por–DNA conjugates. It was found that the Por dimer on the electrode, which was designed to resemble the special pair in natural photosynthesis, shows efficient photocurrent generation in the presence of electron-acceptor reagents compared with the Por monomer. These findings strongly support the idea that the DNA structures could be useful to construct Por arrays, which is essential for the design of photo- and bio-electronic devices.

We describe the synthesis, binding, and electrochemical properties of ferrocene-conjugated oligonucleotides (Fc-oligos). The key step for the preparation of Fc-oligos contains the coupling of vinylferrocene to 5-iododeoxyuridine via Heck reaction. The Fc-conjugated deoxyuridine phosphoramidite was used in the Fc-oligonucleotide synthesis. We show that thiol-modified Fc-oligos deposited onto gold electrodes possess potential ability in electrochemical detection of DNA base mismatch. (C) 2017 Elsevier Ltd. All rights reserved.

Synthetic small molecules with a binding affinity to mismatched base pairs have been widely studied because of their potential applications in chemotherapeutic drugs and diagnostic tools for the detection of single nucleotide variations. In this paper, a luminescent ligand, a perylenediimide (PO) derivative, which is capable of binding to mismatched base pairs within a DNA duplex and generating a strong fluorescence upon the binding event, was demonstrated. Fluorescence enhancement of PO upon the binding to pyrimidine/pyrimidine mismatches were prominent and reached ca. 100-fold for a C/C mismatched base pair, which was visually observed. This result represents the best performance as a fluorescence turn-on response among the existing mismatch binding ligands.

DNA is considered to be a promising biomolecule as a template and scaffold for arranging and organizing functional molecules on the nanoscale. The construction and evaluation of DNAs containing multiple functional molecules that are useful for optoelectronic devices and sensors has been studied. In this paper we report the efficient incorporation of perylenediimide (PDI) units into DNA by using abasic sites both as binding sites and as reactive sites and the construction of PDI stacks within the DNA structure, accomplished through the preorganization of the PDI units in the hydrophobic pocket within the DNA. Our approach could become a valuable method for construction of DNA/chromophore hybrid structures potentially useful for the design of DNA-based devices and biosensors.

Circularly polarized luminescence (CPL) was observed in pyrene zipper arrays helically arranged on an RNA duplex. Hybridization of complementary RNA strands having multiple (two to five) 2'-O-pyrenylmethyl modified nucleosides affords an RNA duplex with normal thermal stability. The pyrene fluorophores are assembled like a zipper in a well-defined helical manner along the axis of RNA duplex, which, upon 350 nm UV illumination, resulted in CPL emission with pyrene excimer formation. CPL (g(lum)) levels observed for the pyrene arrays in dilute aqueous solution were +2x10(-2)-+3.5 x 10(-2), which are comparable with vertical bar g(lum)vertical bar for chiral organic molecules and related systems. The positive CPL signals are consistent with a right-handed helical structure. Temperature dependence on CPL emission indicates that the stable rigid RNA structure is responsible for the strong CPL signals. The single pyrene-modified RNA duplex did not show any CPL signal.

Multichromophore arrays of bis(2-thienyl)diketopyrrolopyrrole (DPP) and naphthalenediimide (NDI) with two Zn-II-cyclens were constructed using thymidine DNA as a scaffold through the binding of the Zn-II-cyclens with thymine bases. We demonstrate photocurrent generation in a donor-acceptor heterojunction configuration consisting of the DPP (donor) and NDI (acceptor) arrays co-immobilized on an Au electrode. The co-immobilized electrode exhibited good photocurrent responses because of the efficient charge separation between the DPP and NDI arrays. In contrast, an immobilized electrode consisting of randomly assembled DPP-NDI arrays generated no photocurrent response because DPP formed ground-state charge-transfer complexes with NDI in the randomly assembled arrays. Therefore, our approach to generate donor-acceptor heterojunctions based on DNA-multichromophore arrays is a useful method to efficiently generate photocurrent.

We describe a simple and convenient method for the preparation of photoresponsive DNA-modified electrodes using primer extension (PEX) reactions. A naphthalimide derivative was used as the photosensitizer that was attached to the C5-position of 2'-deoxyuridine-5'-triphosphate (dUTPNI). It has been found that dUTPNI is a good substrate for the PEX reactions using KOD Dash and Vent (exo-) enzymes in solutions to incorporate naphthalimide (NI) moieties into the DNA sequences. On the electrode surface immobilized with the primer/template DNA, the PEX reactions to incorporate dUTPNI molecules into the DNA sequence were found to efficiently proceed. With this solid-phase method, the DNA monolayers capable of generating photocurrent due to the photoresponsive NI molecule can be constructed. It was shown that the photocurrent generation was significantly suppressed by a single-nucleotide mismatch included in the primer/template DNA, which is applicable for the design of photoelectrochemical sensors to discriminate single-nucleotide sequences.

The charge-transfer process in noncovalent perylenediimide (PDI)/DNA complexes has been investigated by using nanosecond laser flash photolysis (LFP) and photocurrent measurements. The PDI/DNA complexes were prepared by inclusion of cationic PDI molecules into the artificial cavities created inside DNA. The LFP experiments showed that placement of the PDI chromophore at a specific site and included within the base stack of DNA led to the efficient generation of a charge-separated state with a long lifetime by photoexcitation. When two PDI chromophores were separately placed at different positions in DNA, the yield of the charge-separated state with a long lifetime was dependent upon the number of A-T base pairs between the PDIs, which was explained by electron hopping from one PDI to another. Photocurrent generation of the DNA-modified electrodes with the complex was also dependent upon the arrangement of the PDI chromophores. A good correlation was obtained between observed charge separation and photocurrent generation on the PDI/DNA-modified electrodes, which demonstrated the importance of the defined arrangement and assembly of organic chromophores in DNA for efficient charge separation and transfer in multichromophore arrays.

Here we study the binding behavior of perylenediimide (PDI) derivatives to a hydrophobic pocket created inside DNA and their photochemical properties capable of designing a light-up fluorescent sensor for short single-stranded DNA or RNA. The perylenediimide derivative with alkoxy groups (PO) suppressing electron transfer quenching was examined. The PO bound randomly to DNA showed negligible fluorescence due to the aggregation-induced quenching, whereas the PO bound to the pocket as a monomeric form showed more than 100-fold fluorescence enhancement. Switching the binding states of the PO corresponded to a change in the fluorescence response for the hybridization event, which allowed us to design a fluorescent sensor of nucleic acids with a nanomolar detection limit.

Bis(2-thienyl)diketopyrrolopyrrole with two Zn-II-cyclens (ZnCyc-DPP) was designed and synthesized to evaluate the selective binding of Zn-II-cyclen with thymine base in single-strand DNA as a tool for the construction of a highly ordered multichromophore system on DNAs. Through UV/Vis titrations, gel filtration chromatography, and circular dichroism spectroscopy, ZnCyc-DPP formed J-type DPP aggregates with oligo-dT(n) DNAs. The DPP aggregates absorbed on a gold electrode exhibited good photocurrent responses. The present results show that binding Zn-II-cyclen-chromophore conjugates and thymine bases together is a powerful tool for preparing DNA-templated multichromophoric systems with specific functions.

DNA molecules possessing multiple ferrocene (Fc) molecules as a redox active probe were prepared by the primer extension (PEX) reaction using a 2'-deoxyuridine-5'-triphosphate derivative in which Fc was connected to the C5-position of the uridine by a diethylene glycol linker. Gold nanoparticles (AuNP) covered with DNA possessing the Fc molecules were prepared by the PEX reaction on the surface. The AuNP-FcDNA conjugates exhibit a detectable electrochemical signal due to the Fc molecules. Possible application of the PEX reaction on AuNP is demonstrated for the detection of a single nucleotide mutation in the target DNA. (C) 2014 Elsevier Ltd. All rights reserved.

We now report the photocurrent generation and charge transfer dynamics of stacked perylenediimide (PDI) molecules within a pi-stack array of DNA. The cofacially stacked PDI dimer and trimer were found to strongly enhance the photocurrent generation compared to an isolated PDI monomer. Femtosecond time-resolved transient absorption experiments revealed that the excitation of the stacked PDI dimer and trimer provided the broad transient absorption band, which was attributed to the charge delocalization of a negative charge over the PDI chromophores. The lifetime of the charge delocalization of the PDI dimer and trimer (nearly 1 no) was much longer than that of the charge separated state of the PDI monomer. A comparison between the photocurrent measurements and time-resolved transient absorption measurements demonstrated that the cofacially stacked structure could possibly lead to the charge delocalization and increase the lifetime of the charge-separated state that is essential to enhancing the photocurrent generation.

Fluorescent nucleic acids were prepared utilizing the polymerase extension (PEX) reaction to incorporate fluorescent molecules. 2'-Deoxyuridine triphosphate (dUTP) derivatives possessing pyrene molecules as fluorophores were synthesized using the aqueous-phase Sonogashira coupling between 5-Iodo-dUTP and acetylene-linked pyrene molecules. The incorporation of the pyrene (Py)-labeled deoxyuridine triphosphates (PyU) into DNA by polymerase was evaluated by polyacrylamide gel electrophoresis, demonstrating that the PyU can work as a good substrate for the PEX reaction. The fluorescent properties of the functionalized DNA prepared by the PEX reaction were characterized by steady-state fluorescence measurements. The Py-conjugated DNA showed typical emission spectra of the pyrene, and the DNA with two pyrene molecules connected to each other by a diethylene glycol linker exhibited a broadened emission attributed to the electronic interaction between the Py molecules.

We now show that a hydrophobic cavity created within the DNA can work as a scaffold to form a charge transfer (CT) complex composed of naphthalenediimide (NDI) and dialkoxynaphthalene (DAN) derivatives. The formation of the CT complex resulted in stabilization of the duplex structure through stacking interaction, which was comparable to the natural base pairs.

Bis(2-thienyl)-diketopyrrolopyrrole having two $Zn^{II}$-cylcens (DPPCy) was synthesized. DPP-aggregates were constructed by self-organization of DPPCy and $dT_n$-DNAs. In the presence of L-ascorbic acid as an electron sacrifice reagent, the DPP aggregates immobilized on a gold electrode exhibit good anodic photocurrent responses as well as cathodic photocurrent responses in the presence of methyl viologen. The anodic photocurrent responses depend on the DNA lengths because of the formation of uniform DPP-aggregates corresponding to the DNA lengths. The present results show that photocurrent responses of the DPP-aggregates can be controlled by DNA lengths and electron sacrifice reagents.

Small ligand molecules, which can recognize thermodynamically unstable site within DNA, such as mismatch base pair, abasic site, and single-bulge, have attracted much attention because of their potential diagnostics and biological applications. In this paper, we describe the binding of cationic perylenediimide (cPDI) molecules to thymine-containing mismatch base pair in DNA and the formation of cPDI dimer at the mismatch site. The cPDI dimer exhibits a characteristic excimer emission at 650 nm. For T/T mismatch containing DNA, the switching behavior from the PDI dimer (650 nm) to the monomer (550 nm) emission in specific response to Hg2+ ion was observed. (c) 2013 Elsevier Ltd. All rights reserved.

DNA oligonucleotides possessing naphthalimide (NI) at the C5 position of deoxyuridine through an ethynyl-containing linker have been synthesized. Based on time-resolved laser flash photolysis, we showed that the photo-induced charge transfer occurs between NI at the C-5 position and the guanine base of DNA with almost same efficiency (1.8%) when compared to the terminal NI-modified DNA (2.3%). Photoelectrochemical experiments showed that DNA with NI immobilized on the gold electrode generated the photocurrent (28 +/- 2 nA/cm(2)). These results revealed that the NI chromophore located in the extrahelical position could work as a good photosensitizer to induce the charge transfer in DNA. (C) 2013 Elsevier Ltd. All rights reserved.

We prepared various chromophores containing hydrogen bonding unit and investigated their change in photoluminescent properties induced by mechanical stimuli. We reported photoluminescent color and lifetime tuning of complex compound of dyes and acids.

DNA is a unique yet useful material to organize nanoscale molecular arrays along the helix axis. In this study, we demonstrate a useful approach for creating molecular arrays inside a double helical DNA. Our approach is based on a hostguest system. Introducing abasic sites into DNA afforded a hydrophobic cavity that serves as a host. A planar aromatic molecule (cationic perylenediimide, PDI) was used as the guest molecule. In an aqueous solution, the PDI molecules tend to aggregate with themselves due to the strong hydrophobicity. In the presence of DNA with the cavity, the binding of the PDI was found to site-specifically occur in the hydrophobic cavity. The unique assembly and arrangement for more than two PDI molecules was achieved by controlling the sizes and positions of the cavities. Our approach would provide a simple and convenient way to construct one-dimensional aromatic arrays in DNA.

Reversible addition-fragmentation chain transfer (RAFT) polymerization is used to synthesize diblock and random copolymers of poly[(2-trifluoroethyl methacrylate)-co-(methacrylic acid)]. Copolymer films doped with fluorene derivatives comprised of pyridine groups exhibit reversible photoluminescent (PL) color tuning by adjusting the location of fluorene derivatives in H-bonds or non-H-bonds environments. The composite films exhibit green and blue photoluminescence with and without effective H-bonding between the pyridine ring in the fluorene derivative and the carboxylic acid side group, respectively. At elevated temperatures, the H-bonded structure dissociates and the fluorene derivative migrates to the hydrophobic block in the diblock polymer, and they do not form H-bonds upon cooling. However, re-formation of H-bonds also occurs upon cooling random copolymers. Moreover, adjusting the annealing temperature and the solvent annealing reversibly tunes the PL color in the diblock copolymer composite films.

DNA electronic devices were prepared on silicon-based three-terminal electrodes. Both ends of DNA molecules (400 bp long, mixed sequences) were bridged via chemical bonds between the source-drain nanogap (120 nm) electrodes. S-Shaped I-V curves were obtained and the electric current can be modulated by the gate voltage. The DNA molecules act as semiconducting p-type nanowires in the three-terminal device.

DNA oligomers possessing a 2-nitrobenzyl (NB) protected thiol group have been prepared. The photo-remove of the NB to generate a free thiol group in DNA has been analyzed by using reverse-phase HPLC and denaturing gel electrophoresis. The photo-triggered generation of the thiol function in DNA was applicable in the light-initiated ligation of thiol-modified DNA oligomers and Au-DNA conjugation. (C) 2011 Elsevier Ltd. All rights reserved.

p-Stacked naphthalenediimide (NDI) arrays are of interest as charge-transport materials. We have designed and synthesized an NDI derivative with two ZnIIcyclens that act as receptors for the thymine base in DNA. UV/Vis and CD spectroscopy, gel filtration, and molecular-modeling studies have shown that the bis(ZnIIcyclen)NDI can be assembled in the presence of oligo-dT to form p-stacked NDI arrays. The assembly of the NDI arrays was found to be dependent on the length of the oligo-dT and the temperature. The NDIoligo-dT assembly on a gold substrate exhibits photocurrent responses due to electron transfer through the p-stacked array.

The binding and fluorescence properties of complementary RNA sequences attached to different numbers of pyrenes via one carbon linker at the 2'-O-positions have been investigated. Upon hybridization of the pyrene-modified RNA sequences, the modified RNA duplexes with normal thermal stability are formed, and the pyrene arrays are assembled in an inter-strand manner. Because hypochromic effects in the pyrene absorption band and the exciton coupled circular dichroism signals were observed for the pyrene assemblies, the formation of the pyrene array occurs via a pi-stacking interaction between the pyrene rings. The pyrene assemblies exhibit strong excimer fluorescence that is characterized by a broad and structureless excitation spectrum. Hence, the excimer is a static excimer due to the direct excitation of the associated pyrenes in the ground state. Based on several spectroscopies, it is revealed that the spatial configuration of the pyrenes in the association is more regulated by the increase in the attached pyrene.

Electron transfer (ET) through RNA duplexes possessing 2'-O-pyrenylmethy uridine (Upy) and 5-bromouracil (BrU) as an electron donor and accepter set was investigated. Reductive decomposition of the BrU resulted from the ET over long distances (up to ten AU base pairs) was detected in the RNA conjugates. The RNA mediated ET from the pyrene to BrU showed dual distance dependence. This is well consistent with the previous observation for ET from Upy to nitrobenzene in RNA. In contrast, little or no reductive decomposition of the BrU was observed in the DNA conjugates when the Upy and BrU were separated by more than four AT base pairs. (C) 2011 Elsevier Ltd. All rights reserved.

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.

We prepared RNA duplexes possessing 2'-O-(1-pyrenylmethyl)adenosine and 5-(4-nitrophenyl)uridine base pairs. In the duplexes, pyrene serves as a photo-excitable electron donor and 5-(4-nitrophenyl)uridine acts as an electron acceptor. The donor-acceptor-modified RNA duplexes showed very weak fluorescence originating from the pyrene monomer and excimer emissions, which Occur due to electron transfer from the excited pyrene to the nitrobenzene acceptor. (C) 2010 Elsevier Ltd. All rights reserved.

The DNA base stack provides unique features for the efficient long-range charge transfer. For the purpose of investigating excess electron transfer process through DNA, we developed a new method for fluorescence analysis of excess electron transfer based on reductive cleavage of a disulfide bond and a thiol-specific fluorescent probe. Excess electron transfer was detected by monitoring the fluorescence of emissive pyrene monomer generated by the reaction of pyrene maleimides with the cleaved disulfide bond (thiols). Mechanism of reductive cleavage of disulfides through excess electron transfer and subsequent reaction with the fluorescent probes were discussed. This facile and sensitive detection by fluorescence method can be applied for mechanistic study of excess electron transfer. (c) 2009 Elsevier Ltd. All rights reserved.

We describe the long-range excess electron transfer through RNA duplexes consisting of a pyrene electron donor and a nitrobenzene electron acceptor that shows double exponential distance dependence.

RNA molecules with multiple pyrenylmethyl substituents on the 2'-O-sugar residues can form duplexes with complementary RNA sequences without losing thermal stability. In the RNA duplexes, covalently incorporated pyrenes can assemble in a helical manner along the minor grooves of the duplex. These helically assembled pyrene arrays exhibit intense excimer emissions that are efficiently quenched with methyl viologen. (C) 2009 Elsevier Ltd. All rights reserved.

A new approach to electronic detection of a single base mismatch is described. The assay involves the electrochemical measurements of DNA strand exchange reactions (SERs) between electrode-bound redox-modified DNA duplex and target DNA, where the sequence of redox-modified DNA is exchangeable to that of the target DNA. The presence of a single base mismatch can be determined from the slower SER rates compared with fully matched DNA. (c) 2009 Elsevier Ltd. All rights reserved.

Deoxyuridine triphosphate derivatives modified with pyrene was synthesized to functionalize DNA with fluorescent molecules based on the template DNA sequence. Incorporation of pyrene-labeled deoxyuridine triphosphates into DNA by DNA polymerase was investigated by using reverse-phase HPLC and polyacrylamide gel electrophoresis. The fluorescent properties of functionalized DNA were characterized by the steady-state fluorescence measurements.

Template-directed ligation of oligonucleotides by photochemical reaction has attracted much interest because of its biomedical and synthetic applications. In this study, we developed photoligaton of DNA by using a photoremovable protecting group and thiol-disulfide exchange reaction. A phosphoroamidite of o-nitrobenzyl derivatives were synthesized, and DNA modified with a nitrobenzyl-protected thiol group and disulfide group was synthesized by conventional phosphoroamidite chemistry using a DNA synthesizer. It was shown that photochemical reaction of a nitrobenzyl group with UV irradiation produced a free thiol group, leading to the DNA ligation through the thiol-disulfide exchange reaction.

We describe a new ferrocene-based redox-reporter that electronically couples to the DNA Π-electron system.

We synthesized multiple pyrene-modified RNA sequences having two kinds of consecutive sequences, UPyUPy and APyAPy, and investigated their duplex formations and the pyrene associations.

We describe a new strategy for multipyrene modification of RNA sequences to form unique structures of pyrene aromatic arrays, the "pyrene-zipper array", on duplex RNA that exhibits remarkably strong excimer fluorescence.

We describe a general strategy for electronic aptamer-based biosensor that is based on target-induced strand release (TISR) of a redox-modified aptamer from the aptamer-capture DNA duplex bound on an electrode.

A new bis-pyrene-labeled oligonucleotide probe (BP-probe) has been designed for the detection of a single base mismatch in single strand (ss) DNA as a target. The sequence of BP-probe was chosen to form stem-loop structure similar to a molecular beacon (MB-probe), yielding bis-pyrene-labeled molecular beacon (BP-MB-probe). Partially double stranded (ds) BP-MB-probes were prepared by complexation with oligonucleoticles whose sequences are complementary to the loop segment but not to the stem and exchangeable with the target DNA. The partially ds BP-MB-probes were shown to exhibit monomer fluorescence as major fluorescence, while the ss BP-MB-probe in the stem-loop form displays strong excimer fluorescence. The strand exchange reactions between partially ds BP-MB-probe and target ss DNA in the presence of cationic comb-type copolymer as a catalyst were monitored by the excimer fluorescence changes. The existence of a mismatched base can be determined by the slower PASE rates compared with fully matched DNA. (C) 2007 Elsevier Ltd. All rights reserved.

We previously prepared the oligonucleotides (ODNs) conjugated to an anthraquinone (AQ) group via one carbon linker at the 2'-sugar position. When these modified ODNs bind to cDNA sequences, the AQ moiety can be intercalated into the predetermined base-pair pocket of a duplex DNA. In this paper, 2'-AQ-modified ODNs are shown to be an excellent electrochemical probe to clarify the effect of a mismatch base on the charge transfer (CT) though DNA. Two types of DNA-modified electrodes were constructed by assembly of disulfide-terminated 2'-AQ-ODN duplexes onto gold electrodes. One type of electrodes (system I) contains fully matched base pairs or a single-base mismatch in duplex DNA between the redox center and the electrode. The other (system II) consists of the mismatch but at the outside of the redox center. The modified electrodes were analyzed by cyclic voltammetry to estimate the CT rate through duplex DNA. In system I, the CT rate was found to be similar to 50 s(-1) for the fully matched AQ-ODN duplexes, while the CT rates of the mismatched DNA were considerably slower than that of the fully matched DNA. In system II, the AQ-ODN duplexes showed almost similar CT rates (similar to 50 s(-1)) for the fully matched DNA and for the mismatched DNAs. The detection of a single-base mismatch was then performed by chronocoulometry (CC). All the DNA duplexes containing a mismatch base in system I gave the reduced electrochemical responses when compared to the fully matched DNA. In particular, the mismatched DNAs including G-A mismatch can be differentiated from fully matched DNA without using any electrochemical catalyst. We further tested the usefulness of single-stranded (ss) AQ-ODN immobilized on a gold electrode in the electrochemical detection of a single-base mismatch through hybridization assay. The ss-AQ-ODN electrodes were immersed in target-containing buffer at room temperature, and the CC measurements were carried out to see the changes in the integrated charge. Within 60 min, the mismatched DNA was clearly distinguishable by the CC differences from the fully matched target. Thus, the electrochemical hybridization assay provides an easy and convenient detection for DNA mutation that does not require any extra reagents, catalyst, target labeling, and washing steps.

We describe a new strategy for multi-pyrene-modification of RNA sequences to form a unique structure of pyrene arrays on duplex RNA that exhibits remarkably strong excimer fluorescence.

We studied the RNA oligomers having pyrenylmethyl substituents at the 2′-O-sugar residues. It has been shown that the pyrene-modified RNAs can form duplexes with complementary RNA sequences without loss of thermal stability. Absorption, fluorescence, and circular dichroism spectra revealed that the multiply incorporated pyrenes are projected toward the outside of RNA duplexes and assembled in helical aromatic arrays along the minor grooves of the RNA duplexes. The helical pyrene arrays exhibited remarkably strong excimer fluorescence. In the present work, we carried out the fluorescence quenching of the excimer derived from the pyrene-arrays assembled on RNA duplexes in order to evaluate the mobility of the exciton along the pyrene array.