Chikara Dohno

Discrimination of Single-Nucleotide Alterations by G-Specific Fluorescence Quenching

A new strategy for the detection of single‐base alterations through fluorescence quenching by guanine (G) is described. We have devised a novel base‐discriminating fluorescent (BDF) nucleoside, 4′PyT, that contains a pyrenecarboxamide fluorophore at the thymidine sugar’s C4′‐position. 4′PyT‐containing oligodeoxynucleotides only exhibited enhanced fluorescence in response to the presence of a complementary adenine base. In contrast, the fluorescence of mismatched duplexes containing 4′PyT/N base pairs (N=C, G, or T) was considerably weaker. This highly A‐selective fluorescence was a product of guanine‐specific quenching efficiency; when the complementary base to 4′PyT was a mismatch, the pyrenecarboxamide fluorophore was able to interact intimately with neighboring G bases (the most likely interaction in the case of intercalation), so effective quenching by the G bases occurred in the mismatched duplexes. In contrast, duplexes containing 4′PyT/A base pairs exhibited strong emission, since in this case the fluorophores were positioned in the minor groove and able to escape fluorescence quenching by the G bases.

Control of DNA hybridization by photoswitchable molecular glue.

Hybridization of DNA is one of the most intriguing events in molecular recognition and is essential for living matter to inherit life beyond generations. In addition to the function of DNA as genetic material, DNA hybridization is a key to control the function of DNA-based materials in nanoscience. Since the hybridization of two single stranded DNAs is a thermodynamically favorable process, dissociation of the once formed DNA duplex is normally unattainable under isothermal conditions. As the progress of DNA-based nanoscience, methodology to control the DNA hybridization process has become increasingly important. Besides many reports using the chemically modified DNA for the regulation of hybridization, we focused our attention on the use of a small ligand as the molecular glue for the DNA. In 2001, we reported the first designed molecule that strongly and specifically bound to the mismatched base pairs in double stranded DNA. Further studies on the mismatch binding molecules provided us a key discovery of a novel mode of the binding of a mismatch binding ligand that induced the base flipping. With these findings we proposed the concept of molecular glue for DNA for the unidirectional control of DNA hybridization and, eventually photoswitchable molecular glue for DNA, which enabled the bidirectional control of hybridization under photoirradiation. In this tutorial review, we describe in detail how we integrated the mismatch binding ligand into photoswitchable molecular glue for DNA, and the application and perspective in DNA-based nanoscience.

p-Cyano substituted 5-benzoyldeoxyuridine as a novel electron-accepting nucleobase for one-electron oxidation of DNA

Abstract p -Cyano substituted 5-benzoyl-2′-deoxyuridine 1 was synthesized as a novel electron-accepting nucleobase. DNA cleavage by 1 under photoirradiation conditions occurred selectively at the 5′-G of 5′GG3′ sequences after hot piperidine treatment. Photoirradiation of 1 in the presence of dG produced imidazolone as a major product. The electron-accepting nucleobase 1 was successfully incorporated into DNA oligomer by automated DNA synthesis using phosporamidite 2 .

Bidirectional Control of Gold Nanoparticle Assembly by Turning On and Off DNA Hybridization with Thermally Degradable Molecular Glue

Hybridization of single-stranded DNA (ssDNA) with its complementary strand is important not only in many biological processes but also in DNA-based bioassays and nanodevices. The high sequence specificity of hybridization has also been applied to the construction of nanometer-scale structures and DNA-based molecular machines that include controlled motion of DNA. Modulation and control of the formation of double-stranded DNA (dsDNA) is a general strategy for enhancing, limiting, or triggering these biological processes and functions. Several methods have been reported for the control of DNA hybridization by using photochemical and electronic reactions on chemically modified DNA. We have introduced the concept of “molecular glue” for the mediation of hybridization of unmodified DNA with synthetic small molecules that bind to sequences containing up to three contiguous base mismatches. Molecular glue can adhere to two unmodified ssDNA molecules that do not hybridize with each other. The function of the molecular glue that we previously reported relied on thermodynamically favorable ligand binding to two ssDNA molecules. Its function did not include the denaturation of the duplex once formed, and was limited to the unidirectional control of DNA hybridization. To achieve bidirectional control, that is, to turn DNA hybridization on and off, we have looked for methods to inactivate the molecular glue in the reaction system. Here, we describe bidirectional control of DNA hybridization by using the new thermally degradable molecular glue, TD (Scheme 1). Irreversible thermal conversion of TD into its inactive form allowed us to control the DNA hybridization/denaturation cycle. This method for turning DNA hybridization on and off was then applied to the control of the assembly of gold nanoparticles. TD consists of two naphthyridine heterocycles that recognize guanine and selectively bind to dsDNA molecules with G– G mismatches. A key feature of TD, compared to the molecular glue previously reported, is its ability to function as a glue for DNA at room temperature but then be inactivated at an elevated temperature. In order to acquire inactivation, we modified the structure of the original molecule and found that, at an elevated temperature, the bond that connects the two naphthyridine heterocycles can be removed, which remarkably results in the inactivation of the glue function. Thermolysis of TD in 10 mm sodium cacodylate buffer (pH 7.0), as monitored by reversed-phase HPLC, showed that TD was completely consumed within 4 min incubation at 80 8C with concomitant formation of two products (Figure 1). The products were identified as 2-amino-7-methyl-1,8-naphthyridine (1) and (7methyl-1,8-naphthyridin-2-yl)-carbamic acid 2-(2-oxo-oxazolidin-3-yl)-ethyl ester (2 ; Figure 1B). The formation of 1 and 2 indicated that TD underwent thermal fragmentation by an intramolecular carbamate exchange. These results showed that TD could be irreversibly transformed to 1 and 2 by simple heating, and was therefore no longer effective in stabilizing dsDNA. The function of TD as a molecular glue for DNA was confirmed by measuring the melting temperature (Tm) of 11-mer duplexes (5’-d(CTAACXGAATG)-3’/5’-d(CATTCYGTTAG)-3’) that contained mismatches. Experiments were carried out with all possible combinations of matched and mismatched base pairs (represented by X–Y in the sequences) in the absence and presence of TD. TD selectively bound to the dsDNA that contained the G–G mismatch (X=Y=G; see the Supporting Information). In the presence of 100 mm TD, the Tm increase (DTm) for the duplex that contained the G–G mismatch was 25.7 8C; [a] Dr. T. Peng, Dr. C. Dohno, Prof. K. Nakatani Department of Regulatory Bioorganic Chemistry The Institute of Scientific and Industrial Research (SANKEN) Osaka University 8–1 Mihogaoka, Ibaraki 567-0047 (Japan) Fax: (+81)6-6879-8459 E-mail : nakatani@sanken.osaka-u.ac.jp Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. A) The structure of TD and its hydrogen-bonding pattern with guanines on opposing strands of a DNA duplex. B) Schematic representation of TD-assisted hybridization of two ssDNA molecules that contain the sequences 5’-XGG and 5’-XGG (X=C or T), and denaturation of TD-bound dsDNA by heat-induced inactivation of TD followed by gradual cooling to room temperature. The X bases in the XGG/XGG sequence are expected to be flipped-out of the helix, as shown on the right-hand side of the arrow.

The effect of linker length on binding affinity of a photoswitchable molecular glue for DNA.

Molecular glue for DNA is a small synthetic ligand that adheres two single-stranded DNAs to produce a double-stranded DNA. We previously devised a photoswitchable molecular glue (PMG) that uses external light stimuli to reversibly control DNA hybridization. To optimize the structure of PMG, we synthesized a series of PMGs and evaluated the effect of changing the methylene linker length on the binding affinity and photoresponse. From the comprehensive T(m) and CSI-TOF-MS measurements, a PMG possessing a three-methylene linker with carbamate linkage produced maximum binding affinity and photoswitching ability. These results indicate that a small difference in the linker can significantly affect PMG function. These findings are useful for designing new photoswitchable DNA-binding ligands.

Naphthyridine tetramer with a pre-organized structure for 1:1 binding to a CGG/CGG sequence

A naphthyridine carbamate dimer (NCD) is a synthetic ligand for DNA containing a CGG/CGG sequence. Although NCD can bind selectively and tightly to a CGG/CGG sequence, the highly cooperative 2:1 binding mode has hampered precise analysis of the binding. We describe herein the synthesis of a series of naphthyridine tetramers consisting of two NCD molecules connected with various linkers to seek a ligand that binds to a CGG/CGG sequence exclusively with a 1:1 stoichiometry. Among the tested ligands, NCTB and Z-NCTS, which have linker moieties with restricted conformational flexibility [biphenyl and (Z)-stilbene linker, respectively], gave the exclusive formation of a 1:1 ligand–CGG/CGG complex. The (Z)-stilbene linker in Z-NCTS was designed to have pre-organized conformation appropriate for the binding and, in fact, resulted in the highest binding affinity. Thermodynamic parameters obtained by isothermal titration calorimetry indicated that the stronger binding of Z-NCTS was attributed to its lower entropic cost. The present study provides not only a novel 1:1 binding ligand, but also valuable feedback for subsequent molecular design of DNA and RNA binding ligands.

p-Cyano substituted benzophenone as an excellent photophore for one-electron oxidation of DNA

Abstract Novel DNA-photocleaving molecule 1 consisting of a p -cyano substituted benzophenone photophore and an alkyl amino side chain was developed. DNA cleavage by 1 under photoirradiation conditions occurred selectively at 5′ side G of 5′GG3′ sequence after hot piperidine treatment. Photoirradiation of deoxyguanosine at 312 nm in the presence of 1 efficiently produced imidazolone as a predominant product.

Highly selective fluorescent nucleobases for designing base-discriminating fluorescent probes*

There is increasing interest in single nucleotide polymorphism (SNP) typing since they can be used as markers to identify the genes that underlie complex diseases and to realize the full potential of pharmacogenomics in analyzing variable response to drugs. Among the different methodologies for SNP genotyping, the homogenous assay is more amenable than the heterogeneous one. In this article, we will describe some of our most recently developed novel base-discriminating fluorescent (BDF) nucleosides useful for homogenous SNP typing. Our novel concept led to the investigation of a new type of pyrene-labeled BDF nucleosides PyU, PyC, 8pyA, and MePydA, which emitted strong fluorescence only when the bases opposite the BDF bases are A, G, T, and C, respectively. The DNA probes containing four different BDF bases enabled us to distinguish single-base alterations by simply mixing with a sample solution of target DNA. An example of SNP typing of c-Ha-ras SNP sequence has also been demonstrated. Detection of base insertion in insertion/deletion (indel) polymorphisms using pyrene excimer fluorescent probe has also been explored.

Modulation of binding properties of amphiphilic DNA containing multiple dodecyl phosphotriester linkages to lipid bilayer membrane

DNA is a promising functional molecule to modify and design lipid membrane functions. In order to use DNA in a hydrophilic-hydrophobic interface including lipid membrane, we have developed an amphiphilic DNA having dodecyl phosphotriester linkages (dod-DNA). Herein, we report the binding of a series of amphiphilic dod-DNAs to the lipid bilayer membrane. Surface plasmon resonance (SPR) assay and fluorescent microscopy showed that dod-DNA having three dodecyl groups at each end strongly bound to lipid membrane due to the slow dissociation rate and the dod-DNA can be used as a linear template for molecular arrangement on the membrane surface.

Formation of a ligand-assisted complex of two RNA hairpin loops.

The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non-structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand-assisted formation of loop-loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G-G mismatches in double-stranded DNA, we successfully demonstrated the formation of both inter- and intra-molecular NCT6-assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)3 in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly-labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6-assisted loop-loop complex. Förster resonance energy-transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.