Shinsuke Sando

Scanning of guanine-guanine mismatches in DNA by synthetic ligands using surface plasmon resonance

Here we have designed and synthesized ligands that specifically bind with high affinity (Kd = 53 nM) to the guanine (G)–guanine mismatch, one of four types of single-nucleotide polymorphism (SNP). Detection of the G-G mismatch was performed by a surface plasmon resonance (SPR) assay using a sensor chip carrying the G-G specific ligand on its surface. The accuracy of the G-G mismatch detection by the SPR sensor was demonstrated by a marked SPR response obtained only for the DNA containing the G-G mismatch. DNAs containing G-A and G-T mismatches, as well as a fully matched duplex, produced only a weak response. Furthermore, this assay was found applicable for the detection of SNP existing in PCR amplification products of a 652-nucleotide sequence of the HSP70-2 gene.

Light-up Hoechst-DNA aptamer pair: Generation of an aptamer-selective fluorophore from a conventional DNA-staining dye

We have designed a strategy to generate a light‐up fluorophore–aptamer pair based on a down‐modification of a conventional DNA‐staining dye to suppress its affinity to the original dsDNA targets, followed by reselection of aptamers that would bind to the modified dye. Following this line, we prepared a micropolarity‐sensitive Hoechst derivative possessing two tBu groups with low affinity to the usual AT‐rich dsDNA targets. DNA aptamers selected in vitro from a random pool worked as triggers to enhance the fluorescence of an otherwise nonfluorescent Hoechst derivative, and the shortened 25‐mer sequence showed remarkable enhancement (light‐up). The 25‐mer sequence was split into binary aptamer probes, thus enabling us to detect a target nucleic acid sequence with a single‐nucleotide resolution by use of unmodified DNA as a probe.

Cell Surface-Anchored Fluorescent Aptamer Sensor Enables Imaging of Chemical Transmitter Dynamics

A fluorescent aptamer sensor was applied to the analysis of extracellular chemical transmitter dynamics. We utilized a tocopherol-labeled aptamer, which allowed the direct anchoring of the fluorescent aptamer on the cell surface while retaining its performance as a fluorescent sensor. The fast-responsive fluorescent DNA aptamer sensor, which targets adenine compounds, was anchored on the surface of brain astrocytes. Fluorescence imaging of the aptamer-anchored astrocytes enabled the real-time monitoring of release of adenine compounds as a gliotransmitter, which was synchronized with calcium wave propagation in neighboring cells.

A platform for designing hyperpolarized magnetic resonance chemical probes

Hyperpolarization is a highly promising technique for improving the sensitivity of magnetic resonance chemical probes. Here we report [15N, D9]trimethylphenylammonium as a platform for designing a variety of hyperpolarized magnetic resonance chemical probes. The platform structure shows a remarkably long 15N spin–lattice relaxation value (816 s, 14.1 T) for retaining its hyperpolarized spin state. The extended lifetime enables the detection of the hyperpolarized 15N signal of the platform for several tens of minutes and thus overcomes the intrinsic short analysis time of hyperpolarized probes. Versatility of the platform is demonstrated by applying it to three types of hyperpolarized chemical probes: one each for sensing calcium ions, reactive oxygen species (hydrogen peroxide) and enzyme activity (carboxyl esterase). All of the designed probes achieve high sensitivity with rapid reactions and chemical shift changes, which are sufficient to allow sensitive and real-time monitoring of target molecules by 15N magnetic resonance.

Design of chemical shift-switching 19F magnetic resonance imaging probe for specific detection of human monoamine oxidase A.

Monoamine oxidase (MAO) A is a flavoenzyme that catalyzes the oxidation of biologically important monoamines and is thought to be associated with psychiatric disorders. Here, we report a strategy for rationally designing a (19)F magnetic resonance imaging probe for the specific detection of human MAO-A (hMAO-A) activity. Our designed (19)F probe was oxidized expeditiously by hMAO-A to produce 2-fluoro-4-nitrophenol via a spontaneous β-elimination mechanism. Concomitant with the structural change of the probe to the product, the (19)F chemical shift changed by 4.2 ppm, which was enough to visualize the probe and enzymatic product separately. Importantly, our probe achieved excellent discrimination of hMAO-A from its isoform hMAO-B.

Specific binding of 2-amino-1,8-naphthyridine into a single guanine bulge as evidenced by photooxidation of GG doublet

Photoirradiation of 2-amino-1,8-naphthyridines in the presence of duplex DNA containing the GG doublet opposite a single bulge was examined. After hot piperidine treatment, DNA cleavage was observed preferentially at the GG opposite a single bulge. The cleavage efficiency was highly dependent on the nature of bulged base. The G cleavage at the GG opposite a single G bulge was exceptionally weak, suggesting an intercalative binding of 2-amino-1,8-naphthyridine chromophore into the GG step.

Oligonucleotide‐Based Mimetics of Hepatocyte Growth Factor

Oligonucleotide-based hepatocyte growth factor (HGF) mimetics are described. A DNA aptamer to Met, a cognate receptor for HGF, was shown to induce Met activation when used in dimer form. The most potent aptamer dimer, ss-0, which was composed solely of 100-mer single-stranded DNA, exhibited nanomolar potency. Aptamer ss-0 reproduced HGF-induced cellular behaviors, including migration and proliferation. The present work sheds light on oligonucleotides as a novel chemical entity for the design of growth factor mimetics.

Design of a 15 N Molecular Unit to Achieve Long Retention of Hyperpolarized Spin State

Nuclear hyperpolarization is a phenomenon that can be used to improve the sensitivity of magnetic resonance molecular sensors. However, such sensors typically suffer from short hyperpolarization lifetime. Herein we report that [15N, D14]trimethylphenylammonium (TMPA) has a remarkably long spin–lattice relaxation time (1128 s, 14.1 T, 30 °C, D2O) on its 15N nuclei and achieves a long retention of the hyperpolarized state. [15N, D14]TMPA-based hyperpolarized sensor for carboxylesterase allowed the highly sensitive analysis of enzymatic reaction by 15N NMR for over 40 min in phophate-buffered saline (H2O, pH 7.4, 37 °C).

Direct Monitoring of γ-Glutamyl Transpeptidase Activity In Vivo Using a Hyperpolarized (13) C-Labeled Molecular Probe.

The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin.

Design of a Hyperpolarized Molecular Probe for Detection of Aminopeptidase N Activity

Aminopeptidase N (APN) is an important enzyme that is involved in tumor angiogenesis. Detection of APN activity can thus lead to early diagnosis and elucidation of tumor development. Although some molecular probes for APN have been developed, the detection of APN activity in opaque biological samples remains a challenge. To this end, we designed a hyperpolarized NMR probe [1-(13) C]Ala-NH2 which satisfies the prerequisites for APN detection, namely, sufficient retention of the hyperpolarized state, a high reactivity to APN, and an APN-induced chemical shift change. The [1-(13) C]Ala-NH2 probe allowed sensitive detection of APN activity using (13) C NMR spectroscopy.