Clear Distinction of Purine Bases on the Complementary Strand by a Fluorescence Change of a Novel Fluorescent Nucleoside

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

Osmium Complexation of Mismatched DNA: Effect of the Bases Adjacent to Mismatched 5-Methylcytosine

The efficiency of osmium complex formation at 5-methylcytosine in mismatched DNA duplexes is a key point for the design of sequence-specific detection of DNA methylation. Osmium complexation was not observed in fully matched duplexes, whereas the complexation site and efficiency in mismatched duplexes changed depending on the type of 5′-neighboring base of the 5-methylcytosine forming a mismatched base pair. In particular, when the base adjacent to the 5′ side of the mismatched base pair was thymine, a unique “side reaction” was observed. However, the nature of the mismatched base pairs in the reaction site did not influence the selectivity of osmium complex formation with methylated DNA

Nile Red Nucleoside: Design of a Solvatofluorochromic Nucleoside as an Indicator of Micropolarity around DNA

The fluorophore, Nile Red, effectively works as a polarity-sensitive fluorescence probe. We have designed a new nucleoside modified by Nile Red for examining the change in the polarity of the microenvironment surrounding DNA. We synthesized a Nile Red nucleoside (1), formed by replacing nucleobases with Nile Red, through the coupling of a 2-hydroxylated Nile Red derivative and 1,2-dideoxyglycan. This nucleoside showed a high solvatofluorochromicity. The fluorescence of 1 incorporated into DNA was greatly shifted to shorter wavelength by the addition of β-cyclodextrin. The photophysical function of the Nile Red nucleoside will be a good optical indicator for monitoring the change in the micropolarity properties at a specific site on target sequences with interaction between DNA and DNA-binding molecules

Monitoring DNA Structures by Dual Fluorescence of Pyrene Derivatives

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

Intracerebral Distribution of CAG Repeat-Binding Small Molecule Visualized by Whole-Brain Imaging

Understanding the pharmacokinetics of drug candidates of interest in the brain and evaluating drug delivery to the brain are important for developing drugs targeting the brain. Previously, we demonstrated that a CAG repeat-binding small molecule, naphthyridine-azaquinolone (NA), resulted in repeat contraction in mouse models of dentatorubral–pallidoluysian atrophy and Huntington’s disease caused by aberrant expansion of CAG repeats. However, the intracerebral distribution and drug deliverability of NA remain unclear. Here, we report three-dimensional whole-brain imaging of an externally administered small molecule using tissue clearing and light sheet fluorescence microscopy (LSFM). We designed and synthesized an Alexa594-labeled NA derivative with a primary amine for whole-brain imaging (NA-Alexa594-NH2), revealing the intracerebral distribution of NA-Alexa594-NH2 after intraparenchymal and intracerebroventricular administrations by whole-brain imaging combined with tissue clearing and LSFM. We also clarified that intranasally administered NA-Alexa594-NH2 was delivered into the brain via multiple nose-to-brain pathways by tracking the time-dependent change in the intracerebral distribution. Whole-brain imaging of small molecules by tissue clearing and LSFM is useful for elucidating not only the intracerebral distribution but also the drug delivery pathways into the brain

Charge-Pairing Mechanism of Phosphorylation Effect upon Amyloid Fibrillation of Human Tau Core Peptide

Phosphorylation of a fibrillogenic protein, human tau, is believed to play crucial roles in the pathogenesis of Alzheimer’s disease. For elucidating molecular mechanisms of the phosphorylation effect on tau fibrillation, we synthesized a peptide, VQIVY310K (PHF6) and its phosphorylated derivative (PHF6pY). PHF6 is a partial peptide surrounding a plausible in vivo phosphorylation site Tyr310 and forms amyloid-type fibrils similar to those generated by full-length tau. Fibrillation of PHF6 and PHF6pY were studied by spectroscopic and microscopic methods, and the critical concentration of the fibrillation was determined for comparing the fibril stability. The results showed that the phosphorylation strongly influenced the fibrillation propensity of PHF6 by changing its dependency on pH and ionic strength. On the basis of the observations, we suggested that charged sites on the phosphate group and its electrostatic pairing with the neighboring charged residues were physical origins of the phosphorylation effect. To verify this charge-pairing mechanism, we conducted experiments using a series of PHF6 derivatives with non-native charge distributions. The electrostatic interaction in an intermolecular mode was also demonstrated by the system composed of two different peptide species, which found that fibrillation of nonphosphorylated PHF6 was drastically enhanced when a trace amount of phosphorylated PHF6 molecules coexisted. A simulation analysis utilizing crystal coordinates of the PHF6 fibril was also performed for interpreting the experimental results in a molecular level. The present study using the model peptide system gave us a microscopically insightful view on the roles of tau phosphorylation in amyloid-related diseases

Sequence Dependence of Excess Electron Transfer in DNA

DNA-mediated charge transfer has recently received a substantial attention because of its biological relevance in the DNA damage and DNA repair as well as the potential applications to nanoscale electronic devices. In contrast to the numerous mechanistic studies on oxidative hole transfer (HT) through DNA, our understanding of reductive electron transfer process still remains limited. In this article, we demonstrate the results of direct observation of the excess electron transfer (EET) through DNA, which conjugated with aminopyrene (APy) and diphenylacetylene (DPA) as a photosensitizing donor and an acceptor of excess electron, respectively. By inserting dihydrothymine as a spacer between APy and T or C, the yield of electron arrival to DPA was improved. It was indicated that EET through DNA completed within a few or a few tens nanosecond time scale even for EET over 34 Å for both consecutive T and C sequences. The various factors such as mismatch sequence and DNA length on the yield of electron arrival to DPA were examined

Positional Effects of Phosphorylation on the Stability and Morphology of Tau-Related Amyloid Fibrils

Hyperphosphorylated forms of tau protein are the main component of paired helical filaments (PHFs) of neurofibrillary tangles in the brain of Alzheimer’s disease patients. To understand the effect of phosphorylation on the fibrillation of tau, we utilized tau-derived phosphorylated peptides. The V₃₀₆QIVYK₃₁₁ sequence (PHF6) in the microtubule-binding domain is known to play a key role in the fibrillation of tau, and the short peptide corresponding to the PHF6 sequence forms amyloid-type fibrils similar to those generated by full-length tau. We focused on the amino acid residue located at the N-terminus of the PHF6 sequence, serine or lysine in the native isoform of tau, and synthesized the PHF6 derivative peptides with serine or lysine at the N-terminus of PHF6. Peptides phosphorylated at serine and/or tyrosine were synthesized to mimic the possible phosphorylation at these positions. The critical concentrations of the fibrillation of peptides were determined to quantitatively assess fibril stability. The peptide with the net charge of near zero tended to form stable fibrils. Interestingly, the peptide phosphorylated at the N-terminal serine residue exhibited remarkably low fibrillation propensity as compared to the peptide possessing the same net charge. Transmission electron microscopy measurements of the fibrils visualized the paired helical or straight fibers and segregated masses of the fibers or heterogeneous rodlike fibers depending on the phosphorylation status. Further analyses of the fibrils by the X-ray fiber diffraction method and Fourier transform infrared spectroscopic measurements indicated that all the peptides shared a common cross-β structure. In addition, the phosphoserine-containing peptides showed the characteristics of β-sandwiches that could interact with both faces of the β-sheet. On the basis of these observations, possible protofilament models with four β-sheets were constructed to consider the positional effects of the serine and/or tyrosine phosphorylations. The electrostatic intersheet interaction between phosphate groups and the amino group of lysine enhanced the lateral association between β-sheets to compensate for the excess charge. In addition to the previously postulated net charge of the peptide, the position of the charged residue plays a critical role in the amyloid fibrillation of tau

Construction of plasmid from two PCR-amplified DNA fragments.

<p>(A) Scheme of plasmid construction. (B) Primer sequences used for PCR. The two sequences underlined in red and blue are complementary to each other. (C–G) Pictures of agarose gel electrophoresis. (C) PCR-amplified DNA fragments 1.5 (lane 2) and 2.2 kbp (lane 3). (D) 1.5 and 2.2 kbp DNA fragments before (lane 2,3) and after DNA cleavage at 25°C for 48 h (lane 4,5), 37°C for 10 h (lane 6,7), and 70°C for 0.5 h (lane 8,9). MeNH₂ was removed from the samples by speed-vac before electrophoresis. (E) Hybridized 1.5 and 2.2 kbp DNA fragments derived from those without cleavage reaction (lane 2) and cleaved at 25°C for 48 h (lane 3), 37°C for 10 h (lane 4), and 70°C for 0.5 h (lane 5). (F,G) Intact purified plasmids (F) and EcoRV-digested plasmids (G) derived from the DNA fragments cleaved at 25°C for 48 h (lane 2,3), 37°C for 10 h (lane 4–6), and 70°C for 0.5 h (lane 7–9). (H) Sequencing results of primer-derived regions of the plasmids. Underlined letters correspond to EU in the primers.</p

PRODAN-Conjugated DNA: Synthesis and Photochemical Properties

A solvatochromic fluorophore, PRODAN, has been used as a microenvironment-sensitive reporter. Based on the chemistry of PRODAN, we designed and synthesized four novel fluorescent nucleosides, PDNX (X = U, C, A, and G), to which a PRODAN fluorophore was attached at pyrimidine C5 or purine C8. The fluorescent nucleosides sensitively varied the Stokes shift values depending on the orientational polarizability of the solvent. The PDNX incorporated into DNA also changed the Stokes shift values depending on the DNA structure. In particular, the excitation spectrum of the PDNX-containing duplex shifted to a longer wavelength and gave a smaller Stokes shift value when the base opposite PDNX could form a Watson−Crick base pair with PDNX. A lower energy excitation of PDNX-containing DNA resulted in a strong fluorescence emission selective to the Watson−Crick pairing base. This unique photochemical character was applicable to the efficient typing of single-nucleotide polymorphisms of genes