Nucleic acids research. Supplement (2001)最新文献

A new approach was developed for the synthesis of 5'-(Z)-substituted 4',5'-unsaturated adenosines, potential inhibitors against S-adenosyl-L-homocysteine hydrolase (SAHase). A highly stereoselective radical-mediated sulfur-extrusive stannylation was employed as a key step in the present approach. Inhibitory activity of the compounds against SAHase is also presented.

An effective and simple technology is required to identify genes that function in a particular phenotype of interest. A system for identification of genes with libraries of randomized ribozymes has a great potential to investigate gene functions and cellular pathways. This system has been used to identify genes that are relevant to several phenotypes. In vivo application of the library of ribozymes in mice model was also performed to identify genes involved in metastasis. Libraries of ribozymes might be used in vitro and in vivo to study various aspects of basic cell biology and disease processes including metastasis.

New 2',3'-cis-diol protecting groups of methylated G-capping reagents required for the solid-phase synthesis of capped oligonucleotide derivatives were designed so as to have a phenylborate skeleton with an alkylaminomethyl substituent and a DMTr group. The former can stabilize the boronic ester function. The latter led to significant enhancement of the solubility of the capping building units and the spectrometric assay of the DMTr cation generated upon acid treatment of capped products on solid supports enabled us to estimate easily the coupling yield of the 5'-capping reaction. The utility of these protecting groups are discussed.

By conjugating pyrrole (Py)/imidazole (Im) hairpin polyamides with CBI, a stable alkylating moiety, we have developed a new type of DNA-sequence-specific alkylating agent. The sequence specificity and alkylation efficiency of the CBI conjugates were analyzed with high-resolution denaturing gel electrophoresis using linear 450- and 1000-bp DNA. The results demonstrate that CBI conjugates selectively alkylate adenine in specific sequences, whereas the previous type of alkylating polyamide, which contains segment A of DU-86, alkylates both adenine and guanine.

MagiProbe have been demonstrated to be a homogeneous type of probe that emits fluorescence upon hybridization and with specificity for one-base mismatch. To obtain more comprehensive information for the properties of mismatch recognition, examination of mismatch recognition of MagiProbe for the eight single-base mismatches in variety of sequence contexts was performed. The results indicate that the recognition of base mismatches is influenced predominantly by combination of mispaired bases.

Pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy) and 5'-Ru(II) complex labeled oligodeoxyribonucleotie (Ru-probe) were prepared as the fluorescence probes to detect acceptor sites of antisense molecules on native folded RNA under the physiological condition. OMUpy showed the remarkable increase of the fluorescence intensity (334-fold at 375 nm) only when hybridized with complementary oligoRNA. When OMUpy was applied to E. coli 16S-rRNA, the fluorescence intensities were increased in a sequence specific manner. The rotational correlation time of Ru-probe complementary to 16S-rRNA were largely dependent on the sequence of Ru-probe. These results suggest that the accessibility of the antisense molecules for RNA can be evaluated by those fluorescent probes.

Two types of ferrocenyl oligonucleotides, Fc1-ODN and Fc2-ODN, carrying a 5'-ATT GCT CAG GGG TAA GGT CAT TAG TTG GAA-3' sequence were allowed to hybridize with the DNA probe carrying a complementary sequence immobilized on the gold electrode to give rise to a redox signal deriving from their ferrocenyl moieties. When a mixture of these oligonucleotides in a proper ratio was used as a model of DNA samples, two redox peaks were observed in one differential pulse voltammogram. The peak currents varied depending on the initial ratio of the two oligonucleotides, suggesting a possibility that this technique may be applied to electrochemical monitoring of gene expression by, for example, the electrochemical differential hybridization, EDH.

Over 15 years ago, the Benner group noticed that the DNA alphabet need not be limited to the four standard nucleotides known in natural DNA. Rather, twelve nucleobases forming six base pairs joined by mutually exclusive hydrogen bonding patterns are possible within the geometry of the Watson-Crick pair (Fig. 1). Synthesis and studies on these compounds have brought us to the threshold of a synthetic biology, an artificial chemical system that does basic processes needed for life (in particular, Darwinian evolution), but with unnatural chemical structures. At the same time, the artificial genetic information systems (AEGIS) that we have developed have been used in FDA-approved commercial tests for managing HIV and hepatitis C infections in individual patients, and in a tool that seeks the virus for severe acute respiratory syndrome (SARS). AEGIS also supports the next generation of robotic probes to search for genetic molecules on Mars, Europa, and elsewhere where NASA probes will travel.

A linear, covalently-closed, dumbbell-shaped DNA vector including a transcription unit is known to have both biological stability and safety and is expected to be useful for gene therapy. We established an easy, quick, and large preparative synthetic method of modified- and unmodified-dumbbell DNA using an intramolecular cyclization at the DNA termini.

An efficient and practical synthesis of 4'-thioribonucleosides was accomplished via the Pummerer reaction. The resulting 4'-thioribonucleosides were converted into the corresponding phosphoramidite units, and 4'-thioribonucleic acids (4'-thioRNAs) of 15 mer were synthesized on a DNA synthesizer using a controlled pore glass (CPG) with 4'-thiouridine unit. The thermal stability of 4'-thioRNA:RNA duplex was higher than that of RNA:RNA duplex. Moreover, the thermal stability of 4'-thioRNA:4'-thioRNA duplex was the highest and stabilized 30 degrees C or more compared with RNA:RNA duplex (>99 degrees C vs 66 degrees C). Structural analysis by CD spectra indicates that 4'-thioRNA:4'-thioRNA duplex and 4'-thioRNA:RNA duplex adapt A-form conformation as well as RNA:RNA duplex.