Biomedical subjects
Publications and source records attributed to Akimitsu Okamoto.
We altered the fluorescence emission selectivity of a base-discriminating fluorescent base, (Py)U, from A-selective to T-selective by the addition of mercury ion. The strong fluorescence from a duplex containing the (Py)U/T base pair was specific to the mercury ion among divalent metal ions, providing a unique method for sensing mercury ions in aqueous solutions.
We have developed a nucleotide modified by a pyrene derivative with dual fluorescence. The dual fluorescence of the fluorophore, which was incorporated into DNA, was effectively controlled at ambient temperature according to DNA structural status. Our nucleoside with dual fluorescence is effective as a conceptually new probe for monitoring DNA hybridization by the color change without multilabeling with fluorescent dyes.
Novel public-key system using DNA has been developed. To solve key distribution problem, the public-key cryptography system based on the one-way function has been developed. The message-encoded DNA hidden in dummies can be restored by PCR amplification, followed by sequencing. We used these operations as a one-way function, and constituted a novel method for the key distribution based on the public-key system using DNA. We will show the way of holding a key in common just between specific two persons.
We have developed a new fluorescent DNA sensor containing two pyrene-labeled nucleobases, (Pet)G and (Py)C, and the fluorescence color was altered by the salt-induced B-Z DNA transition.
Photostimulated hole transport through DNA duplexes immobilized on gold electrodes has been investigated. By modifying a gold electrode with a DNA duplex containing a photosensitizer, we have observed a sequence-dependent cathodic photocurrent. DNA acts as a good mediator for cathodic photocurrent when appropriate sequences are selected.
We report on the enzymatic synthesis of DNA wires by successive incorporation of artificial nucleotides. We synthesized an efficient hole-transporting nucleoside triphosphate, d(MD)ATP, and have examined the screening of polymerases and reverse transcriptases for enzymatic incorporation of d(MD)ATP into DNA. We found that KOD Dash is an effective enzyme for the enzymatic synthesis of (MD)A-containing DNA. Furthermore, the efficiency of enzymatic incorporation was remarkably improved by the addition of manganese chloride. By applying the PCR method to the synthesis of DNA containing a run of (MD)A, the (MD)A run was amplified up to 6 x 10(6) times. (MD)A runs prepared by the enzymatic method exhibited a high hole-transporting ability.
A conceptually new logic gate based on DNA has been devised. Methoxybenzodeazaadenine ((MD)A), an artificial nucleobase which we recently developed for efficient hole transport through DNA, formed stable base pairs with T and C. However, a reasonable hole-transport efficiency was observed in the reaction for the duplex containing an (MD)A/T base pair, whereas the hole transport was strongly suppressed in the reaction using a duplex where the base opposite (MD)A was replaced by C. The influence of complementary pyrimidines on the efficiency of hole transport through (MD)A was quite contrary to the selectivity observed for hole transport through G. The orthogonality of the modulation of these hole-transport properties by complementary pyrimidine bases is promising for the design of a new molecular logic gate. The logic gate system was executed by hole transport through short DNA duplexes, which consisted of the "logic gate strand", containing hole-transporting nucleobases, and the "input strand", containing pyrimidines which modulate the hole-transport efficiency of logic bases. A logic gate strand containing multiple (MD)A bases in series provided the basis for a sharp AND logic action. On the other hand, for OR logic and combinational logic, conversion of Boolean expressions to standard sum-of-product (SOP) expressions was indispensable. Three logic gate strands were designed for OR logic according to each product term in the standard SOP expression of OR logic. The hole-transport efficiency observed for the mixed sample of logic gate strands exhibited an OR logic behavior. This approach is generally applicable to the design of other complicated combinational logic circuits such as the full-adder.
Novel ODN probes for insertion polymorphism detection have been devised. The ODN probes labeled with two pyrene chromophores emit strong excimer fluorescence when hybridized with DNA containing one inserted base. This method is useful for the determination of the presence/absence of insertion polymorphisms located at a specific site on the target DNA.
We report on a new method for the detection of a base at a specific site in a DNA sequence by monitoring the fluorescence emission of fluorescein. To achieve this goal, we developed a new base-discriminating fluorescent (BDF) nucleobase, naphthodeazaadenine ((ND)A). The fluorescence spectrum of the duplex possessing a cytosine base as a complementary base of (ND)A showed a fluorescence peak at 383 nm when using an excitation wavelength of 350 nm. When the complementary base of (ND)A was one of the other bases, the fluorescence intensity was very low. The fluorescence emission spectrum of (ND)A overlapped with the fluorescence excitation spectrum of fluorescein in the wavelength range of 400-500 nm. Thus, we designed FRET-BDF probes containing (ND)A as the FRET donor and fluorescein as the acceptor. The interaction of these two fluorophores, which are separated by defined base pairs, allowed an efficient energy transfer that resulted in a dominant fluorescence emission of fluorescein at 520 nm when using an excitation wavelength of 350 nm. Fluorescence emission from FRET-BDF probes was observed only when the complementary base of (ND)A is C, thus achieving a clear distinction of a C base on the complementary DNA strand. However, the general utility of our method is limited due to the quenching of the (ND)A fluorescence by a G/C base pair flanking (ND)A.
A guanine derivative with a covalently linked nitroxide spin label has been devised. The spin label was incorporated into oligodeoxynucleotides by post-synthetic modification. The local environment of a variety of G-rich DNA is detectable by ESR using this spin probe.
A novel fluorescence BDF probe containing pyrene-labeled 7-deaza-2[prime or minute]-deoxyadenosine has been developed for the detection of thymine base on a target DNA.
This paper describes the design of novel base-discriminating fluorescent (BDF) nucleobases and their application to single nucleotide polymorphism (SNP) typing. We devised novel BDF nucleosides, (Py)U and (Py)C, which contain a pyrenecarboxamide chromophore connected by a propargyl linker. The fluorescence spectrum of the duplex containing a (Py)U/A base pair showed a strong emission at 397 nm on 327 nm excitation. In contrast, the fluorescence of duplexes containing (Py)U/N base pairs (N = C, G, or T) was considerably weaker. The proposed structure of the duplex containing a matched (Py)U/A base pair suggests that the high polarity near the pyrenecarboxamide group is responsible for the strong A-selective fluorescence emission. Moreover, the fluorescence of the duplex containing a (Py)U/A base pair was not quenched by a flanking C/G base pair. The fluorescence properties are quite different from previous BDF nucleobases, where fluorescence is quenchable by flanking C/G base pairs. The duplex containing the C derivative, (Py)C, selectively emitted fluorescence when the base opposite (Py)C was G. The drastic change of fluorescence intensity by the nature of the complementary base is extremely useful for SNP typing. (Py)U- and (Py)C-containing oligodeoxynucleotides acted as effective reporter probes for homogeneous SNP typing of DNA samples containing c-Ha-ras and BRCA2 SNP sites.
We have developed a novel nucleosbase, edaG, that efficiently releases various reporter units upon one-electron oxidation. The edaG-selective degradation of ODNs was achieved by various mild oxidizing agents. The oxidant-dependent molecular releasing technique is quite useful not only for DNA-based drug releasing systems but also for the detection of long-range hole transport through DNA without time-consuming analysis.
We demonstrated that a P-loop, a looped complex formed inside duplex DNA by adding peptide nucleic acids (PNA), acts catalytically as a template for enzymatic cleavage of single-stranded probe oligodeoxynucleotides (ODN). A PD-loop complex formed from P-loop and probe ODN was digested efficiently by a restriction enzyme, and the truncated probe ODN was released. The P-loop nicked by the enzyme can form PD-loop again with another probe ODN, and then assisted the enzymatic cleavage of an excess of probe ODN. In addition, by using dumbbell-formed ODN as a probe ODN, the efficiency of the P-loop-assisted ODN cleavage was enhanced considerably as compared with that of linear ODN. Thus, the method utilizing P-loop will make it possible to amplify the sequence information of duplex DNA via a catalytic cleavage of probe ODNs.
We report a novel method for base detection using a base-discriminating fluorescent (BDF) nucleoside. We developed BDF probes containing methoxybenzodeazaadenine MDA and methoxybenzodeazainosine MDI, which give strong fluorescence only when the base on the complementary strand is cytosine and thymine, respectively. Thus, the MDA- and MDI-containing ODNs can be used as a very effective BDF probe for the detection of single base alterations, such as SNPs and point mutations. The present method using BDF probes is a very powerful tool for SNP typing that does not require any enzymes and time-consuming steps, and can avoid hybridization errors. In addition, a combination of MDA- and MDI-containing BDF probes facilitates the T/C SNP typing of a heterozygous sample.
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DNA is a promising conductive biopolymer. However, there are problems that need to be solved to realize real DNA wires. These include the low efficiency of hole transport and the serious oxidative damage that can occur during hole transport. We have demonstrated a protocol for the design of a DNA wire that can effectively mediate hole transport that is not adversely affected by oxidation during hole transport through the DNA duplex. We have synthesized a stable and effective DNA wire by incorporating a designer nucleobase, benzodeazaadenine derivatives, which have lower oxidation potentials and wider stacking areas but are not decomposed during hole transport.
A new fluorescent nucleoside, benzopyridopyrimidine (BPP), which can sharply distinguish between A and G bases opposite BPP has been devised. The base-pairing degeneracy of BPP strongly contributes to the sharp fluorescence change that is dependent on the type of purine bases opposite BPP. The hybridization of an ODN probe containing BPP with a target DNA facilitates the judgment with the naked eye of the type of purine base located at a specific site on the target DNA. The BPP-containing ODN is a very effective probe for A/G SNP typing.