Nucleic acids research. Supplement (2001)最新文献

Development of peptide/protein synthesis is very important for investigating the functions of peptides and proteins. The advantages of cell-free synthesis, which has been extensively developed, include the ability to synthesize toxic proteins, aggregated proteins and proteins containing unnatural amino acids. However, this promising approach does not have a high reaction yield. In this study, we examine whether the rolling synchronization system, which produces long RNAs with a repeated sequence encoded by very small circular ssDNAs without the promoter sequence for RNA polymerase, can be used for cell-free peptide synthesis to improve the reaction yield. As a result, we have found that rolling synchronization is useful for large-scale cell-free peptide/protein synthesis.

Alkaliphilic Bacillus sp. strain 41M-1 produces an alkaliphilic xylanase (xylanase J). The newly constructed mutant E177Q deltaJC had an acidic pH optimum and showed almost no activity at pH 8.0. The alkaliphily of the enzyme was restored by directed evolution. The evolved mutants, Y176S/E177Q deltaJC and G32V/Y176D/E177Q deltaJC, retained about 30% and 43% activity of their maximal activities at pH 6.0, respectively.

We have developed an efficient and extensive deuterium incorporation method using a heterogeneous Pd/C-D2O-H2 system into the base moiety of nucleic acids. The results presented here provide a deuterium gas-free, totally catalytic and post-synthetic deuterium labeling method in D2O media.

A marine photosynthetic bacterium Rhodovulum sulfidophilum secretes nucleic acids that are involved in flocculating ability. These extracellular nucleic acids have not been well characterized. Here, we have analyzed these nucleic acids and revealed that the extracellular nucleic acids are a mixture of double-stranded DNAs and single-stranded RNAs. The DNAs have sizes of more than 30 kbp and at least a part of these DNAs is probably an amplified portion of genomic DNA. The RNAs seems to be tRNA like molecules from size estimation.

Previously, we synthesized oligodeoxyribonucleotides (ODNs) containing novel base pairing motif consisting of tricyclic nucleosides, Im-N(O) and Im-O(N), with the ability to form four hydrogen bonds (H-bonds). When the base pair, Im-N(O):Im-O(N) pair, was incorporated into the duplex, thermal stability of the duplex depended on the sequence context. In this time, on the basis of previous results, we designed two new bicyclic nucleosides, Na-N(O) and Na-O(N) as complementary nucleobase of the tricyclic nucleobases. These nucleosides were synthesized via a Pd-mediated cross coupling reaction as a key step. When the new Im-O(N):Na-N(O) pair was incorporated into the duplex, it was remarkably stabilized independent of the sequence context.

3'-Fluoro analogue 1 of selective and potent adenosine A3 receptor agonist, Cl-IB-MECA was synthesized from D-xylose via highly regioselective opening of lyxo-epoxide 4 with fluoride anion. Compared to the high binding affinity of Cl-IB-MECA to the A3 adenosine receptor, the corresponding 3'-fluoro derivative showed remarkably decreased binding affinity, indicating that 3'-hydroxyl group acts as hydrogen bonding donor, not hydrogen bonding acceptor like fluorine atom in binding to the A3 adenosine receptor.

Very specific binding of of Hg(II) and Ag(I) cations unexpectedly and significantly stabilizes the naturally occurring miss-base pairs, thymine-thymine and cytosine-cytosine, in DNA duplexes.

We developed a novel synthesis of biologically important cyclic bis(3'-->5')diguanylic acid (cGpGp). The present synthesis includes two strategies different from those employed in an existing synthesis. They are the phosphoramidite method for the preparation of a guanylyl(3'-->5')guanylic acid intermediate and allyl protection for guanine bases and internucleotide linkages. These distinctive strategies have allowed the new synthesis to provide the target compound in a higher yield than that of the existing synthesis.

We designed and successfully synthesized novel nucleic acid analogues with a C3'-C4' trans-fused six-membered ring, trans-3',4'-BNAs from D-glucose. A 1H-NMR experiment and X-ray crystallographic analysis demonstrated that the sugar puckering of trans-3',4'-BNA was restricted in an S-type conformation. We also achieved its incorporation into oligonucleotides using automated DNA synthesizer.

In a search of suitable recognition molecules for the formation of stable non-natural triplex DNA, we have developed the new nucleoside analogs (WNA, W-shape nucleoside analog) bearing an aromatic ring, a recognition base and a bicyclic skeleton to hold them. A successful result with WNA-betaT that recognizes a TA base pair selectively has suggested that the basic structure of the WNA is useful as a scaffold for search of other candidates for the formation of triplex containing a TA or a CG interrupting site. In this study, we have synthesized and investigated binding property of a variety of WNA candidates. As a result, we have determined new potential analog, WNA-betaC for a CG interrupting site. It should be noted that the triplexes containing a WNA-betaT:TA or a WNA-betaC:CG base triplet have exhibited higher stability than the natural anti-parallel triplexes constructed of A:AT and G:GC triplets. The results that H-WNA-betaT lacking a benzene ring did not show stabilization effect to any base pairs have clearly indicated that a benzene ring plays a key role in stability of triplexes.