Structural optimization of pseudorotaxane-forming oligonucleotides for efficient and stable complex formation.

Interlocked structures, such as rotaxane and catenane, combine both static and dynamic properties. To expand their unique properties into the chemical biology field, a spontaneous formation method of the interlocked structures with the target would be ideal. We have previously developed a pseudorotaxane-forming oligo DNA (prfODN) to spontaneously form topological DNA/RNA architectures. In this study, we report the structural optimization of prfODNs for the efficient and stable complex formation. The optimized prfODNs efficiently formed pseudorotaxane structures with a DNA or RNA target, and the yield for the RNA target reached 85% in 5 min. In addition, the optimized prfODNs could form the pseudorotaxane structure with a smaller ring size and the structure significantly increased the kinetic stability. Furthermore, the catenane structure was successfully formed with the optimized prfODNs to provide the conclusive evidence for the formation of the threaded structure. This information will be valuable for developing new chemical methods using functional nucleic acids for antisense oligo nucleotides and DNA/RNA nanotechnology.

Alkylating probes for the G-quadruplex structure and evaluation of the properties of the alkylated G-quadruplex DNA.

The G-quadruplex structure has been found in biologically significant regions of the genomic DNA, including the telomere and promoter regions, and is known to play an important role in a number of biological processes. In this paper, we report the development of alkylating probes for the G-quadruplex structure and evaluation of the properties of the modified G-quadruplex structure.

Selective alkylation of T-T mismatched DNA using vinyldiaminotriazine-acridine conjugate.

The alkylation of the specific higher-order nucleic acid structures is of great significance in order to control its function and gene expression. In this report, we have described the T-T mismatch selective alkylation with a vinyldiaminotriazine (VDAT)-acridine conjugate. The alkylation selectively proceeded at the N3 position of thymidine on the T-T mismatch. Interestingly, the alkylated thymidine induced base flipping of the complementary base in the duplex. In a model experiment for the alkylation of the CTG repeats DNA which causes myotonic dystrophy type 1 (DM1), the observed reaction rate for one alkylation increased in proportion to the number of T-T mismatches. In addition, we showed that primer extension reactions with DNA polymerase and transcription with RNA polymerase were stopped by the alkylation. The alkylation of the repeat DNA will efficiently work for the inhibition of replication and transcription reactions. These functions of the VDAT-acridine conjugate would be useful as a new biochemical tool for the study of CTG repeats and may provide a new strategy for the molecular therapy of DM1.

Pseudorotaxane formation via the slippage process with chemically cyclized oligonucleotides.

Circular nucleic acids have been utilized for versatile applications by taking advantage of the unique characteristic of their circular structure. In our previous study, we found that the chemically-cyclized ODN (cyODN) with double-tailed parts formed a pseudorotaxane structure with the target via the slippage process. We now report the investigation of the slippage properties and the mechanism of the slippage process using six different cyODNs. Our results indicate that the formation efficiency significantly depend on the temperature, the ring size, the target length and the mismatched position of the target. The kinetic studies also showed that this pseudorotaxane formation would proceed via a non-threaded structure which hybridizes with the target at the double-tailed parts. In addition, the resulting pseudorotaxanes showed interesting characteristics unlike the canonical duplex such as the hysteresis loop in the Tm measurements and the kinetic stabilization by lengthening the target. This information will be fundamentally important for finding new functions of circular nucleic acids and elucidating the threading mechanism regarding other synthetic small molecules and biopolymers.

Synthesis of peptide-conjugated light-driven molecular motors and evaluation of their DNA-binding properties.

Synthetic light-driven molecular motors are molecular machines capable of rotation under photo-irradiation. In this paper, we report the synthesis of peptide-conjugated molecular motors and evaluate their DNA-binding properties.

Fast DNA interstrand cross-linking reaction by 6-vinylpurine.

Oligonucleotides that incorporate a reactive moiety to form an interstrand cross-link have been widely studied for their potential toward inhibiting gene expression or as basic tools for chemical biology studies. The 6-vinylpurine (2) newly designed in the current study serves well as a new purine-base moiety for increasing cross-link reactivity to target cytosine. Thus, oligonucleotides containing 6-vinylpurine exhibit a more selective and much smoother DNA cross-linking ability to cytosine than the oligonucleotide analogs derived from 2-amino-6-vinylpurine (1) previously explored.

Green-colored plastids in the dinoflagellate genus Lepidodinium are of core chlorophyte origin.

Most photosynthetic dinoflagellates possess plastids containing chlorophyllsa+c,but species belonging to the genus Lepidodiniumare unique in bearing non-canonical plastids containing chlorophyllsa+b. According to the pioneering works on pigment composition data, it has been proposed that Lepidodiniumplastids were derived from a prasinophyte species, though this hypothesis was not supported by a recent phylogenetic analysis based on an alignment comprised of eight plastid proteins (Takishita et al. 2008, Gene 410: 26-26). This "8-protein" analysis however was insufficient to clarify the origin of Lepidodiniumplastids for two major reasons: First, the alignment lacked sufficient evolutionary information to resolve the precise origin of Lepidodiniumplastids. Second, the taxa considered did not well represent the diversity of Chlorophyta. Particularly, prasinophytes were poorly sampled in the alignment. In this study, we sequenced plastid-encoded genes from L. chlorophorum, one pedinophyte species, one ulvophyte species, and six prasinophyte species. The 85 sequences newly determined in this study and recent progress in plastid genome sequencing enabled us to prepare an alignment comprised of 11 plastid proteins from green algal taxa that appropriately cover the diversity of Chlorophyta. All the analyses of the 11-protein data set robustly grouped L. chlorophorumwith members of the "core chlorophytes." Thus, we here propose that Lepidodiniumplastids are of core chlorophyte origin.