Nucleic acids symposium series (2004)最新文献

We examined the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid (3'-amino-2',4'-BNA) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The melting temperature of the pyrimidine motif triplex at pH 6.8 with 3'-amino-2',4'-BNA modified TFO was significantly higher than that observed with unmodified TFO. The 3'-amino-2',4'-BNA modification of TFO increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the 3'-amino-2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

At least two separate enzymes, an endonuclease and a ligase, are thought to be involved in the tRNA splicing pathway. The yeast and archaeal endonucleases acting in the first step of tRNA splicing commonly produce 2', 3'-cyclic phosphate and 5' hydroxy group at the exon-intron borders. Despite this similarity in the first step of tRNA splicing, the subsequent mechanism of archaeal splicing pathway has not been elucidated yet. We have been searching for the archaeal ligase activity from Methanosarcina acetivorans. Here, we report the distinct activity of a splicing endonuclease detected in its cell extract.

Accumulation of genetic changes due to the presence of unrepaired DNA lesions can lead to cancer development and other diseases. Nucleic acid modifications also play key roles in many essential life processes. We have developed a series of chemically modified nucleic acid analogues that can be applied to stabilize protein-nucleic acid interactions for structural and proteomic studies. Some of the probes have also been employed to study nucleic acid-nucleic acid interactions.

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

Survival strategies of many bacterial pathogens, including Pseudomonas aeruginosa, are linked to their ability to form surface associated communities called biofilms. The biofilm life style allows these organisms to persist in various tissues, avoid clearance by innate host defences and significantly enhanced their resistance to antibiotics. Formation of various biofilm components, including the synthesis of the extracellular polysaccharide matrix, is controlled at the transcriptional and translational levels and also by a small molecule second messenger bis-(3',5')-cyclic-di-guanidine monophosphate (c-di-GMP). The synthesis of c-di-GMP from GTP and its degradation is controlled by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), encoded by over thirty genes in the P. aeruginosa genome. We have shown that an increase in the intracellular c-di-GMP levels favors biofilm formation due to its role as a cofactor for the synthesis of several types of extracellular polysaccharides, including PEL and alginate, the two key virulence factors of P. aeruginosa during infection of patients with cystic fibrosis. During biosynthesis of PEL and alginate, c-di-GMP binds to specific receptors, PelD and Alg44, respectively. We have also recently demonstrated that DGCs have a relaxed specificity and can cyclize other nucleotides besides GTP. These atypical cyclic dinucleotides bind c-di-GMP receptors with high affinity, suggesting that intracellular regulation of various biological functions by this group of second messengers may be more complex than previously recognized.

We examined the effects of molecular crowding conditions on the structures and thermodynamics of three-way junctions (TWJs) of DNA. To explore the crowding effects on the junction point, we further evaluated the number of water molecules associated with the whole TWJ as well as the individual arms. It was found that the number of water molecules taken up by the whole TWJ was significantly smaller than the sum of the individual arms. These results clearly show the dehydration from the junction point of the TWJ structure. Therefore, molecular crowding should be favourable for the junction point of TWJ structure and unfavourable for the duplex structure. From these results, it can be concluded that a cell-mimicking molecular crowding condition in which the activity of water decreases and hydration becomes less favourable, might facilitate the formation of junction structures in comparison with duplexes.

Template-directed ligation of oligonucleotides by photochemical reaction has attracted much interest because of its biomedical and synthetic applications. In this study, we developed photoligaton of DNA by using a photoremovable protecting group and thiol-disulfide exchange reaction. A phosphoroamidite of o-nitrobenzyl derivatives were synthesized, and DNA modified with a nitrobenzyl-protected thiol group and disulfide group was synthesized by conventional phosphoroamidite chemistry using a DNA synthesizer. It was shown that photochemical reaction of a nitrobenzyl group with UV irradiation produced a free thiol group, leading to the DNA ligation through the thiol-disulfide exchange reaction.

PNA (peptide nucleic acid) is a DNA analogue which has a peptide backbone. PNA is very useful as a recognition probe because of its immensely high affinity to DNA. In this study, we used PNA as a DNA separation tool. Furthermore, we introduced a cleavable disulfide bond into main chain of PNA in order to facilitate the removal of PNA from PNA/DNA duplex. This disulfide bond was readily cleaved by various reducing agents and then the resultant two short PNA fragments could not form stable PNA/DNA duplex anymore. Accordingly, a desired DNA fragment was picked up from the DNA mixtures and readily recovered by the reductive treatment.

We theoretically evaluated the stability, UV-Vis spectra and possibility of stacking of [S-M(II)-S] (M=Ni, Pd, Pt, S: hydroxypyridonethione) by means of the density functional theory (DFT). From the view of the free energy, we assessed formation energy of possible combinations of chalcogen atoms and metal cations. The results confirmed that [H-Ni(II)-H] and [S-Cu(II)-S] would form stable metal-base pairing, on the other hand [H-Pt(II)-H] would not, which have been experimentally proven. Moreover, by use of time-dependent density functional theory (TDDFT), we observed d-pi* transition accompanied with pi-pi* transition in [S-M(II)-S]. These results reveal that metal-to-ligand charge transfer (MLCT) shifts the peak of pi-pi* transition in [S-2H(+)-S] (without metal cations).

We have previously demonstrated that heme, iron(III)-protoporphyrin IX complex, and a parallel-quadruplex DNA assembled from d(TTAGGG) form a stable coordination complex called "heme-DNA complex". The heme-DNA complex exhibits a variety of spectroscopic characteristics remarkably similar to those of met-form of myoglobin, oxygen-binding hemoprotein, reflecting that the heme environments in the two systems are highly alike to each other. In a course of our effort toward exploring functional properties of the heme-DNA complex, we have investigated binding of gaseous molecules such as CO and O(2) to the heme-DNA complex. The present study revealed that the heme-DNA complex exhibits ability to accommodate these molecules as exogenous ligands.