Akio Kobori

Surprising repair activities of nonpolar analogs of 8-oxoG expose features of recognition and catalysis by base excision repair glycosylases.

Repair glycosylases locate and excise damaged bases from DNA, playing central roles in preservation of the genome and prevention of disease. Two key glycosylases, Fpg and hOGG1, function to remove the mutagenic oxidized base 8-oxoG (OG) from DNA. To investigate the relative contributions of conformational preferences, leaving group ability, enzyme-base hydrogen bonding, and nucleobase shape on damage recognition by these glycosylases, a series of four substituted indole nucleosides, based on the parent OG nonpolar isostere 2Cl-4F-indole, were tested as possible direct substrates of these enzymes in the context of 30 base pair duplexes paired with C. Surprisingly, single-turnover experiments revealed that Fpg-catalyzed base removal activity of two of the nonpolar analogs was superior to the native OG substrate. The hOGG1 glycosylase was also found to catalyze removal of three of the nonpolar analogs, albeit considerably less efficiently than removal of OG. Of note, the analog that was completely resistant to hOGG1-catalyzed excision has a chloro-substituent at the position of NH7 of OG, implicating the importance of recognition of this position in catalysis. Both hOGG1 and Fpg retained high affinity for the duplexes containing the nonpolar isosteres. These studies show that hydrogen bonds between base and enzyme are not needed for efficient damage recognition and repair by Fpg and underscore the importance of facile extrusion from the helix in its damaged base selection. In contrast, damage removal by hOGG1 is sensitive to both hydrogen bonding groups and nucleobase shape. The relative rates of excision of the analogs with the two glycosylases highlight key differences in their mechanisms of damaged base recognition and removal.

Selective Photo-Cross-Linking of 2′-O-Psoralen-Conjugated Oligonucleotide with Rnas Having Point Mutations

It has been reported that point mutations in genes are responsible for various cancers and the selective regulation of the gene expression is an important issue to develop a new type of anticancer drugs. In this report, we present a new type of antisense molecule that photo-cross-links to an oligoribonucleotide having a point mutation site in a sequence specific manner. 2′-O-psoralen-conjugated adenosine was synthesized in four steps from adenosine and introduced in the middle of an oligodeoxyribonucleotide (2′-Ps-oligo). Compared with 5′-O-psoralen-conjugated oligodeoxyribonucleotide (5′-Ps-oligo), which has a psoralen at the 5′-end, 2′-Ps-oligo more selectively photo-cross-linked to a pyrimidine base of the site of alteration from purine to pyrimidine in the oligoribonucleotide.

Detection of L-DNA-tagged PCR products by surface plasmon resonance imaging.

The employment of tags plays an increasingly important role in genomics, in which PCR products are arrayed at the microor nanoscale. PCR products have been labeled in many ways. One conventional method is to incorporate labeled nucleotides into the PCR products during the extension reaction by DNA polymerase. Another method is to use a PCR primer that is labeled at the 5’ end. PCR primers with fluorophore labels are used for DNA sequencing, whereas biotinylated primers are used for the isolation and detection of the PCR products due to their strong and specific binding to streptavidin. Here, we report on a mirror-image DNA (l-DNA)-tagged PCR (LT-PCR) that enables us to label the PCR products with a defined sequence of l-DNA tag. The important findings leading to LT-PCR were that the l-DNA tag did not interfere with the PCR and remained as a single strand even after PCR. Therefore, LT-PCR products could be precisely delivered onto the DNA microarray, where the l-DNA complementary to the tag sequence was immobilized. A large number of sequences available for lDNA tags would be suitable for such comprehensive microarray analysis. We demonstrated that the surface plasmon resonance imaging (SPR) array carrying the complementary l-DNA on the surface successfully detected the LT-PCR products without any purification or additional fluorescent labeling. l-DNA consisting of l-2’-deoxyriboses is an enantiomer of natural DNA (d-DNA) and has unique properties. l-DNA has been shown to be a poor substrate for the human endoand exo-nucleases, as it has the opposite chirality to their intrinsic target, and does not interact with single-stranded d-DNA, whereas it does bind sequence-selectively to complementary l-DNA. We anticipated that an l-DNA tag attached to a PCR primer at the 5’-end would not be recognized as a PCR template by DNA polymerase and would remain in a single-stranded form after amplification (Scheme 1). Besides l-DNA, a peptide nucleic acid (PNA) or d-DNA tag attached through a nonreplicable linker were conceivable for coding PCR products. However, the binding ability of PNA or d-DNA to complementary d-DNA might induce the formation of undesirable intraprimer hairpins or an interaction to an unexpected sequence of the template DNA. The intrinsic possibility of forming hairpin secondary structures and ambiguity in the effect on the PCR reactions thus makes them unsuitable for the labeling. Primers used in LT-PCR consist of three parts, d-DNA to function as a general PCR primer, a l-DNA unit as a molecular tag, and three l-dTs as a spacer between the d and l-DNA. The spacer was inserted to mitigate steric congestion that might be produced between the left-handed and the right-handed duplexes upon hybridization of LT-PCR products with a com-

Dimer of 2,7-diamino-1,8-naphthyridine for the detection of mismatches formed by pyrimidine nucleotide bases

Discrimination of base mismatches from normal Watson-Crick base pairs in duplex DNA constitutes a key approach to the detection of single nucleotide polymorphisms (SNPs). We have developed a sensor for a surface plasmon resonance (SPR) assay system to detect G-G, A-A, and C-C mismatch duplexes by employing a surface upon which mismatch-binding ligands (MBLs) are immobilized. We synthesized a new MBL consisting of 2,7-diamino-1,8-naphthyridine (damND) and immobilized it onto a CM5 sensor chip to carry out the SPR assay of DNA duplexes containing a single-base mismatch. The SPR sensor with damND revealed strong responses to all C-C mismatches, and sequence-dependent C-T and T-T mismatches. Compared to ND- and naphthyridine-azaquinolone hybrid (NA)-immobilized sensor surfaces, with affinity to mismatches composed of purine nucleotide bases, the damND-immobilized surface was useful for the detection of the mismatches composed of pyrimidine nucleotide bases.

SYNTHESIS AND PROPERTIES OF OLIGODEOXYRIBONUCLEOTIDES CONTAINING 4-N-ACETYLCYTOSINE BASES

Abstract Oligodeoxyribonucleotides containing 4-N-acetyl-2′-deoxycytidines (ac4dC) were synthesized by the H-phosphonate method. Thymidine 3′-O-(3,4-dichloro)phthalate bound to a solid support was employed as the starting material for the solid-phase synthesis. The (3,4-dichloro)phthaloyl (DCP) linker was found to be cleaved by treatment with 10% DBU in CH3CN for 5 min without loss of the 4-N-acetyl group of 2′-deoxycytidine. The thermal stability of the duplexes containing ac4dC was investigated.

Sequence selective formation of 1,N6-ethenoadenine in DNA by furan-conjugated probe

1,N(6)-Ethenoadenosine derivatives have been applied as fluorescence probes in various fields of biochemistry and molecular biology. We developed a 1,N(6)-ethenoadenosine-forming reaction at a target adenine in DNA duplex and applied it to a mutation diagnosis. Furan-derivatized oligodeoxyribonucleotides were synthesized and fluorescence properties were studied in the presence of complementary strand under oxidative conditions. Strong emissions at 430nm were observed in the presence of the complementary strand with an adenine in front of furan moiety.

Novel photoresponsive cross-linking oligodeoxyribonucleotides having a caged α-chloroaldehyde.

We have developed photoresponsive cross-linking oligodeoxyribonucleotides (ODNs) for sequence-selective interstrand covalent bond formation toward target nucleotides. A phosphoramidite derivative of α-chloroaldehyde whose carbonyl group was converted to a bis(2-nitrobenzyl)acetal group was prepared for the synthesis of photoresponsive α-chloroaldehyde (PCA)-conjugated ODN. The bis(2-nitrobenzyl)acetal group of a PCA-thymidine conjugate was completely removed by UV irradiation at 365 nm (400 mW/cm(2)) for 1 min. Photo-cross-linking studies revealed that PCA-ODN selectively reacted with the target nucleotides having an adenine or a cytosine moiety at the frontal position of the α-chloroaldehyde group.

Cross-Linking Antisense Oligodeoxyribonucleotides with a Photoresponsive α-Chloroaldehyde Moiety for RNA Point Mutations.

Because point mutations in GTPase-coding genes have been reported to be responsible for the transformation of cells, anticancer reagents that react effectively and sequence selectively with target RNAs having a point mutation are highly desired. In this study, we developed novel photo-cross-linking oligodeoxyribonucleotides ((pro)PCA-ODNs) that had a caged α-chloroaldehyde group conjugated to a 2-methylpropanediyl backbone ((pro)PCA) in the middle of the strand. A kinetic study of the deprotection reaction of (pro)PCA-ODN revealed that the bis(2-nitrobenzyl)acetal group was completely deprotected within 1 min. Photo-cross-linking studies of (pro)PCA-ODNs with complementary oligoribonucleotides (ORNs) revealed that (pro)PCA-ODNs reacts efficiently and selectively with the target ORNs that have an adenosine or cytidine residue at a frontal position of the (pro)PCA residue without adverse effects of bases adjacent to the mutation site.

Rate-adjusted cross-linking reaction by photoresponsive α-bromoaldehyde (PBA)-conjugated ODN.

We developed a photoresponsive α-bromoaldehyde-conjugated oligonucleotide (PBA-ODN). The PBA-ODN selectively reacted and formed covalent bonds with target oligonucleotides having adenine or cytosine at the frontal position of the aldehyde derivative. Kinetic studies revealed that PBA-ODN has increased kinetic rates for the formation of cross-linked duplexes compared with the corresponding α-chloroaldehyde-conjugated oligonucleotide (PCA-ODN).

RNA-based diagnosis in a multicellular specimen by whole mount in situ hybridization using an RNA-specific probe.

Recent RNA research has revealed the close involvement of various RNAs in cellular functions. RNAs are becoming the inevitable target molecules for research into details of gene expression. RNA and its related complexes are also promising targets for disease diagnosis. Multi cellular specimens such as organ tissues, histopathological specimens, and embryos are among the possible targets of RNA-based diagnostic techniques. In this report, we focused on a method that would provide such spatial and temporal information. We demonstrated that an RNA-specific probe (OMUpy2) was not only applicable to the detection of a specific mRNA in Drosophila embryos in a temporal and spatial manner but was also relatively quick and easy to use. The probe, OMUpy2, could be applied to other multi cellular systems for RNA-based diagnosis and research. The promising results of this manuscript show the great potential of RNA-based detection for both biological research and diagnostic medicine.