Takenori Dairaku

The structure of metallo-DNA with consecutive thymine–HgII–thymine base pairs explains positive entropy for the metallo base pair formation

We have determined the three-dimensional (3D) structure of DNA duplex that includes tandem HgII-mediated T–T base pairs (thymine–HgII–thymine, T–HgII–T) with NMR spectroscopy in solution. This is the first 3D structure of metallo-DNA (covalently metallated DNA) composed exclusively of ‘NATURAL’ bases. The T–HgII–T base pairs whose chemical structure was determined with the 15N NMR spectroscopy were well accommodated in a B-form double helix, mimicking normal Watson–Crick base pairs. The Hg atoms aligned along DNA helical axis were shielded from the bulk water. The complete dehydration of Hg atoms inside DNA explained the positive reaction entropy (ΔS) for the T–HgII–T base pair formation. The positive ΔS value arises owing to the HgII dehydration, which was approved with the 3D structure. The 3D structure explained extraordinary affinity of thymine towards HgII and revealed arrangement of T–HgII–T base pairs in metallo-DNA.

Thermodynamic and structural properties of the specific binding between Ag+ ion and C:C mismatched base pair in duplex DNA to form C–Ag–C metal-mediated base pair

Metal ion-nucleic acid interactions have attracted considerable interest for their involvement in structure formation and catalytic activity of nucleic acids. Although interactions between metal ion and mismatched base pair duplex are important to understand mechanism of gene mutations related to heavy metal ions, they have not been well-characterized. We recently found that the Ag(+) ion stabilized a C:C mismatched base pair duplex DNA. A C-Ag-C metal-mediated base pair was supposed to be formed by the binding between the Ag(+) ion and the C:C mismatched base pair to stabilize the duplex. Here, we examined specificity, thermodynamics and structure of possible C-Ag-C metal-mediated base pair. UV melting indicated that only the duplex with the C:C mismatched base pair, and not of the duplexes with the perfectly matched and other mismatched base pairs, was specifically stabilized on adding the Ag(+) ion. Isothermal titration calorimetry demonstrated that the Ag(+) ion specifically bound with the C:C base pair at 1:1 molar ratio with a binding constant of 10(6) M(-1), which was significantly larger than those for nonspecific metal ion-DNA interactions. Electrospray ionization mass spectrometry also supported the specific 1:1 binding between the Ag(+) ion and the C:C base pair. Circular dichroism spectroscopy and NMR revealed that the Ag(+) ion may bind with the N3 positions of the C:C base pair without distorting the higher-order structure of the duplex. We conclude that the specific formation of C-Ag-C base pair with large binding affinity would provide a binding mode of metal ion-DNA interactions, similar to that of the previously reported T-Hg-T base pair. The C-Ag-C base pair may be useful not only for understanding of molecular mechanism of gene mutations related to heavy metal ions but also for wide variety of potential applications of metal-mediated base pairs in various fields, such as material, life and environmental sciences.

High‐Resolution Crystal Structure of a Silver(I)–RNA Hybrid Duplex Containing Watson–Crick‐like CSilver(I)C Metallo‐Base Pairs

Metallo-base pairs have been extensively studied for applications in nucleic acid-based nanodevices and genetic code expansion. Metallo-base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo-base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T-Hg(II)-T base pairs. Herein, we have determined a high-resolution crystal structure of the second natural metallo-base pair between pyrimidine bases C-Ag(I)-C formed in an RNA duplex. One Ag(I) occupies the center between two cytosines and forms a C-Ag(I)-C base pair through N3-Ag(I)-N3 linear coordination. The C-Ag(I)-C base pair formation does not disturb the standard A-form conformation of RNA. Since the C-Ag(I)-C base pair is structurally similar to the canonical Watson-Crick base pairs, it can be a useful building block for structure-based design and fabrication of nucleic acid-based nanodevices.

Raman spectroscopic detection of the T-HgII-T base pair and the ionic characteristics of mercury

Developing applications for metal-mediated base pairs (metallo-base-pair) has recently become a high-priority area in nucleic acid research, and physicochemical analyses are important for designing and fine-tuning molecular devices using metallo-base-pairs. In this study, we characterized the HgII-mediated T-T (T-HgII-T) base pair by Raman spectroscopy, which revealed the unique physical and chemical properties of HgII. A characteristic Raman marker band at 1586 cm−1 was observed and assigned to the C4=O4 stretching mode. We confirmed the assignment by the isotopic shift (18O-labeling at O4) and density functional theory (DFT) calculations. The unusually low wavenumber of the C4=O4 stretching suggested that the bond order of the C4=O4 bond reduced from its canonical value. This reduction of the bond order can be explained if the enolate-like structure (N3=C4-O4−) is involved as a resonance contributor in the thymine ring of the T-HgII-T pair. This resonance includes the N-HgII-bonded state (HgII-N3-C4=O4) and the N-HgII-dissociated state (HgII+ N3=C4-O4−), and the latter contributor reduced the bond order of N-HgII. Consequently, the HgII nucleus in the T-HgII-T pair exhibited a cationic character. Natural bond orbital (NBO) analysis supports the interpretations of the Raman experiments.

A metallo-DNA nanowire with uninterrupted one-dimensional silver array

The double-helix structure of DNA, in which complementary strands reversibly hybridize to each other, not only explains how genetic information is stored and replicated, but also has proved very attractive for the development of nanomaterials. The discovery of metal-mediated base pairs has prompted the generation of short metal–DNA hybrid duplexes by a bottom-up approach. Here we describe a metallo-DNA nanowire—whose structure was solved by high-resolution X-ray crystallography—that consists of dodecamer duplexes held together by four different metal-mediated base pairs (the previously observed C–Ag–C, as well as G–Ag–G, G–Ag–C and T–Ag–T) and linked to each other through G overhangs involved in interduplex G–Ag–G. The resulting hybrid nanowires are 2 nm wide with a length of the order of micrometres to millimetres, and hold the silver ions in uninterrupted one-dimensional arrays along the DNA helical axis. The hybrid nanowires are further assembled into three-dimensional lattices by interactions between adenine residues, fully bulged out of the double helix. A metallo–DNA hybrid nanowire composed only of silver-mediated base pairs has been prepared and its crystal structure resolved by X-ray diffraction. The nanowire, which is 2 nm wide and whose length reaches the μm to mm scale, holds silver ions into uninterrupted one-dimensional arrays along the DNA helical axis.

Structure Determination of an Ag I -Mediated Cytosine–Cytosine Base Pair within DNA Duplex in Solution with 1 H/ 15 N/ 109 Ag NMR Spectroscopy

The structure of an Ag(I) -mediated cytosine-cytosine base pair, C-Ag(I) -C, was determined with NMR spectroscopy in solution. The observation of 1-bond (15) N-(109) Ag J-coupling ((1) J((15) N,(109) Ag): 83 and 84 Hz) recorded within the C-Ag(I) -C base pair evidenced the N3-Ag(I) -N3 linkage in C-Ag(I) -C. The triplet resonances of the N4 atoms in C-Ag(I) -C demonstrated that each exocyclic N4 atom exists as an amino group (-NH2 ), and any isomerization and/or N4-Ag(I) bonding can be excluded. The 3D structure of Ag(I) -DNA complex determined with NOEs was classified as a B-form conformation with a notable propeller twist of C-Ag(I) -C (-18.3±3.0°). The (109) Ag NMR chemical shift of C-Ag(I) -C was recorded for cytidine/Ag(I) complex (δ((109) Ag): 442 ppm) to completed full NMR characterization of the metal linkage. The structural interpretation of NMR data with quantum mechanical calculations corroborated the structure of the C-Ag(I) -C base pair.

Direct detection of the mercury-nitrogen bond in the thymine-Hg II -thymine base-pair with 199 Hg NMR spectroscopy

We have observed the 1-bond (199)Hg-(15)N J-coupling ((1)J((199)Hg,(15)N) = 1050 Hz) within the Hg(II)-mediated thymine-thymine base pair (T-Hg(II)-T). This strikingly large (1)J((199)Hg,(15)N) is the first one for canonical sp(2)-nitrogen atoms, which can be a sensitive structure-probe of N-mercurated compounds and a direct evidence for N-mercuration.

Exploring a DNA Sequence for the Three-Dimensional Structure Determination of a Silver(I)-Mediated C-C Base Pair in a DNA Duplex By (1)H NMR Spectroscopy.

Recently, we discovered novel silver(I)-mediated cytosine–cytosine base pair (C–AgI–C) in DNA duplexes. To understand the properties of these base pairs, we searched for a DNA sequence that can be used in NMR structure determination. After extensive sequence optimizations, a non-symmetric 15-base-paired DNA duplex with a single C–AgI–C base pair flanked by 14 A–T base pairs was selected. In spite of its challenging length for NMR measurements (30 independent residues) with small sequence variation, we could assign most non-exchangeable protons (254 out of 270) and imino protons for structure determination.

Preparation of Nafion/Polycation Layer-by-Layer Films for Adsorption and Release of Insulin

Thin films were prepared using layer-by-layer (LbL) deposition of Nafion (NAF) and polycations such as poly(allylamine hydrochloride) (PAH), poly(ethyleneimine) (PEI), and poly(diallydimethylammonium chloride) (PDDA). Insulin was then adsorbed on the NAF-polycation LbL films by immersion in an insulin solution. The NAF-polycation LbL films were characterized using a quartz crystal microbalance and an atomic force microscope. The release of insulin from the LbL films was characterized using UV-visible adsorption spectroscopy and fluorescence emission spectroscopy. The greatest amount of insulin was adsorbed on the NAF-PAH LbL film. The amount of insulin adsorbed on the (NAF/PAH)5NAF LbL films by immersion in a 1 mg mL−1 insulin solution at pH 7.4 was 61.8 µg cm−2. The amount of insulin released from the LbL films was higher when immersed in insulin solutions at pH 2.0 and pH 9.0 than at pH 7.4. Therefore, NAF-polycations could be employed as insulin delivery LbL films under mild conditions and as an insulin release control system according to pH change.

Preparation of Microparticles Capable of Glucose-Induced Insulin Release under Physiological Conditions

Hydrogen peroxide (H2O2)-sensitive layer-by-layer films were prepared based on combining phenyl boronic acid (PBA)-modified poly(allylamine) (PAH) with shikimic acid (SA)-modified-PAH through boronate ester bonds. These PBA-PAH/SA-PAH multilayer films could be prepared in aqueous solutions at pH 7.4 and 9.0 in the presence of NaCl. It is believed that the electrostatic repulsion between the SA-PAH and PBA-PAH was diminished and the formation of ester bonds between the SA and PBA was promoted in the presence of NaCl. These films readily decomposed in the presence of H2O2 because the boronate ester bonds were cleaved by an oxidation reaction. In addition, SA-PAH/PBA-PAH multilayer films combined with glucose oxidase (GOx) were decomposed in the presence of glucose because GOx catalyzes the oxidation of D-glucose to generate H2O2. The surfaces of CaCO3 microparticles were coated with PAH/GOx/(SA-PAH/PBA-PAH)5 films that absorbed insulin. A 1 mg quantity of these particles released up to 10 μg insulin in the presence 10 mM glucose under physiological conditions.