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.Download high-res image (50KB)Download full-size image

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.

We now report the photocurrent generation and charge transfer dynamics of stacked perylenediimide (PDI) molecules within a π-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 ns) 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.

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.

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.

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.

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/cm2). 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.

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.

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 π-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.

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.A new method for fluorescence analysis of excess electron transfer through DNA based on reductive cleavage of a disulfide bond and a thiol-specific fluorescent probe is described.

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.RNA duplexes possessing 2′-O-(1-pyrenylmethy)adenosine and 5-(4-nitrophenyl)uridine base pairs were prepared. The RNA duplexes showed very weak fluorescence due to pyrene monomer and excimer emissions, which resulted from the electron transfer from the excited pyrene to the nitrobenzene acceptor.

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.

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.Electronic detection of DNA mutation based on DNA strand exchange reactions (SERs) is described.The presence of a single base mismatch can be electrochemically determined from the slower SER rates compared with fully matched DNA.

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 ∼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 (∼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.

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 oligonucleotides 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.

A pyrene–excimer-forming probe allowed the easy and sensitive detection of a single base mismatch in target DNA. This was due to the faster strand exchange rate compared to a fully-matched target.

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 π-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.