Nucleic acids research. Supplement (2001)最新文献

In order to screen and study of RNA aptamers against HCV NS3 helicase domain, we performed in vitro selection using two kinds of RNA pools, N30H and N30V. After eight selection cycles, RNA aptamers obtained from N30H pool possessed 5' extended single-stranded regions and the conserved sequence [5'-GGA(U/C)GGAGCC-3'] at stem-loop regions. On the other hand RNA aptamers obtained from another pool, N30V, possessed 3' long single-stranded regions with several stem-loop structures. All these aptamers showed strong inhibition of helicase activity in vitro, especially #5 represented Kd = 20 nM and IC50 approximately 50 nM. Interestingly these RNA aptamers showed similar secondary structural configuration with positive-stranded 3' untranslated region [3'(+)UTR]. It is known that 3'UTR of HCV is important for replication of HCV and also has strong affinity with NS3 protein. We will discuss the interaction of NS3 and 3'(+)UTR.

Crystal structures of DNA octamer with the sequence d(GCGAGAGC) have been determined by X-ray analyses to investigate the specific DNA structural motifs that are useful for designing various functional DNA molecules. The octamers are assembled to form an octaplex with G-quartets and water-mediated A-quartets. At relatively high potassium concentration, however, the octaplex is split into two quadruplexes, each of which contains two G-duets. The two crystal forms suggest a dynamic formation of an octaplex from two quadruplexes in solution.

As a part of our recent studies of RNA cleavage using antisense oligonucleotide-metal complex(es) conjugates, we prepared self-complementary 2'-O-methyloligonucleotides with a terpyridine x Cu(II) complex, which was attached to the sugar portion of the 5'-end or 3'-end. The thermal stabilities of the formed duplexes, as compared with those of the unmodified 2'-O-methyl duplexes, revealed that both the 5'- and 3'-complexes greatly enhanced the duplex stability.

We synthesized novel adenosine derivatives tethering an aromatic hydrocarbon group by an amido linker. The single adenosine derivatives at 5' dangling end stabilized the DNA duplex of 5'-ATGCGCAT-3' more or equally than Watson-Crick base pair. When the number of the dangling residues increased from one to three, the duplex stability became larger by 1.8-3.6 kcal/mol. When the adenosine derivative was opposite to an abasic site in a DNA duplex, the destabilization by the abasic site was significantly reduced. These observations suggest that the adenosine derivatives developed in this study can stack with a DNA base pair by forming a base pair-mimic geometry.

DNA binding of the ligand bearing an oxine and a pyridinium group was regulated by coexisting Cu2+ over the binding constant range of three orders of magnitude. The ligands coordinated to Cu2+ to form a dimer and then cooperatively bound outside of DNA duplex to give the well-regulated 1-D structure along the DNA backbone.

In cyanobacterium Synechococcus sp. PCC 7942, SmtB, functioning as the sensor to heavy-metal ions (notably Zn-ion) in the dimer form, represses the transcription of smtA gene encoding metallothionein-like protein. There are two recognition DNA sequences in the operator/promoter region of smtA gene, and the binding of SmtB with Zn-ions reduces the affinities to these sequences. This induces the activation of smtA transcription. In this study, we have studied the functional differences between these two recognition DNA sequences, with spectroscopic (hetero-nuclear NMR and UV-resonance Raman) and biochemical methods. On the basis of the results obtained here, we clarified the regulation mechanism of the smtA expression. Similar regulatory system for the tolerance to Zn-ion stress is found in another cyanobacterium. We also found that the functionally important differences between these two systems are mainly due to the structural differences of recognition DNA sequences.

The long-range interactions between the peripheral domains of Tetrahymena group I intron were studied by NMR. The 20mer RNA contained the 9.1a loop region (AUGCAA) and the 17mer RNA contained the 2.1 loop region (AGAUUGC) were synthesized and studied by NMR. They form both hairpin structures at low NaCl concentration. The 20mer RNA forms dimer by loop-loop interaction, while the 17mer RNA is monomer. On the addition of the 17mer to the 20mer, the new imino proton signals induced by the interaction between the 9.1a and 2.1 loops were not observed. It is found that the 9.1a loop-loop interaction (GCAA-GCAA) with two base pairs prefers to that between the 9.1a and 2.1 loops (GCAA-UUGC) with four base pairs. By my model of intact folding structure containing A-minor tertiary interaction and the compared with sequence, it is presumed that the 9.1a loop does not bind to the 2.1 loop, but to the 5c loop (UGCAA), while the 2.1 loop binds to 3' exon (UAA).

DNA fragments containing the sequence d(GCGAAAGC) adopt a base-intercalated (zipper-like) duplex in crystalline state at high ionic strength. To investigate effects of point mutation at the 5th residue on the structure, two crystal structures of d(GCGAGAGC) and d(GCGATAGC) have been determined by X-ray crystallography. In both crystals, the two octamers related by a crystallographic two-fold symmetry are aligned in an anti-parallel fashion and associated to each other to form a duplex, suggesting that the base-intercalated duplex is stable even when the 5th residue is mutated with other bases. The sheared G3:A6 pair formation makes the two phosphate backbones closer and facilitates formation of the A-X*-X-A* base-intercalated motif. The three duplexes are assembled around the two hexamine cobalt chlorides. The central X and X* residues are bound to a different cation depending on the kinds of bases.

Conjugate DNAzymes were synthesized by solid phase fragment condensation and their biological properties were characterized. They have increased affinity to target RNA, enhanced stability against DNase 1 digestion and comparable or higher RNA cleaving activity compared with native and also phosphorothioate DNA zymes. It was also demonstrated that conjugate DNAzymes could inhibit BCR-ABL tyrosine kinase in cellular lysis of human leukemia cell line. Consequently DNAzymes can be expected to act effectively in cellular system and also in vivo system.

The mutagenicity of oxidized dATP, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), was examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. 2-OH-dATP mainly elicited a G x C to A x T transition, and a G x C to T x A transversion to a lesser extent. Interestingly, the mutagenicity of 2-OH-dATP was enhanced in the presence of 2-hydroxydeoxyadenosine 5'-diphosphate, an inhibitor of the MTH1 protein, suggesting that this protein functions in the hydrolysis of 2-OH-dATP in the replication reaction mixture. These results indicate that 2-OH-dATP is mutagenic and that its mutagenicity is suppressed by the MTH1 protein in mammalian cells.