Uracil-selective Cross-linking in RNA and Inhibition of miRNA Function by 2-Amino-6-vinyl-7-deazapurine Deoxynucleosides.

MicroRNAs (miRNAs) regulate gene expression through RNA interference. Consequently, miRNA inhibitors, such as anti-miRNA oligonucleotides (AMOs), have attracted attention for treating miRNA overexpression. To achieve efficient inhibition, we developed 2-amino-6-vinylpurine (AVP) nucleosides that form covalent bonds with uridine counterparts in RNA. We demonstrated that mRNA cross-linked with AVP-conjugated antisense oligonucleotides with AVP were protected from gene silencing by exogenous miRNA. However, endogenous miRNA function could not be inhibited in cells, probably because of slow cross-linking kinetics. We recently developed ADpVP, an AVP derivative bearing a 7-propynyl group-which boasts faster reaction rate than the original AVP. Here, we synthesized dADpVP-a deoxy analog of ADpVP-through a simplified synthesis protocol. Evaluation of the cross-linking reaction revealed that the reaction kinetics of dADpVP were comparable to those of ADpVP. In addition, structural analysis of the cross-linked adduct discovered N3 linkage against uridine. Incorporating dADpVP into two types of miRNA inhibitors revealed a marginal impact on AMO efficacy yet improved the performance of target site blockers. These results indicate the potential of cross-linking nucleosides for indirect miRNA function inhibition.

Selective alkylation of parallel G-quadruplex structure.

The selective alkylation of nucleic acids is important for a medicinal approach and biological study. We now report a novel selective alkylation of the parallel G-quadruplex structure using the conjugate of the macrocyclic hexaoxazole L2G2-6OTD-1M1PA and vinyl-quinazolinone-S(O)Me (6OTD-VQ-S(O)Me).

Reactive OFF-ON type alkylating agents for higher-ordered structures of nucleic acids.

Higher-ordered structure motifs of nucleic acids, such as the G-quadruplex (G-4), mismatched and bulge structures, are significant research targets because these structures are involved in genetic control and diseases. Selective alkylation of these higher-order structures is challenging due to the chemical instability of the alkylating agent and side-reactions with the single- or double-strand DNA and RNA. We now report the reactive OFF-ON type alkylating agents, vinyl-quinazolinone (VQ) precursors with a sulfoxide, thiophenyl or thiomethyl group for the OFF-ON control of the vinyl reactivity. The stable VQ precursors conjugated with aminoacridine, which bind to the G-4 DNA, selectively reacted with a T base on the G-4 DNA in contrast to the single- and double-strand DNA. Additionally, the VQ precursor reacted with the T or U base in the AP-site, G-4 RNA and T-T mismatch structures. These VQ precursors would be a new candidate for the T or U specific alkylation in the higher-ordered structures of nucleic acids.

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.

Large-scale analysis of small molecule-RNA interactions using multiplexed RNA structure libraries.

The large-scale analysis of small-molecule binding to diverse RNA structures is key to understanding the required interaction properties and selectivity for developing RNA-binding molecules toward RNA-targeted therapies. Here, we report a new system for performing the large-scale analysis of small molecule-RNA interactions using a multiplexed pull-down assay with RNA structure libraries. The system profiled the RNA-binding landscapes of G-clamp and thiazole orange derivatives, which recognizes an unpaired guanine base and are good probes for fluorescent indicator displacement (FID) assays, respectively. We discuss the binding preferences of these molecules based on their large-scale affinity profiles. In addition, we selected combinations of fluorescent indicators and different ranks of RNA based on the information and screened for RNA-binding molecules using FID. RNAs with high- and intermediate-rank RNA provided reliable results. Our system provides fundamental information about small molecule-RNA interactions and facilitates the discovery of novel RNA-binding molecules.

A promising visible light-driven photocatalytic activity of conjugated polymer nanocrystals.

Conjugated polymers have recently emerged as a new class of photocatalysts. However, many conjugated polymer photocatalysts are not as effective as inorganic materials due to the limited electronic properties of their LUMO and HOMO and low long-term stability caused by the degradation of the conjugated backbone. In the present study, we have demonstrated, for the first time, the superior Visible (Vis) light-driven photocatalytic activity of conjugated polymer nanocrystals (NCs) of polydiacetylene (PDA) derivatives, namely, p-DCHD NCs for the photodegradation of Rhodamine B (RhB) compared with that of the state-of-the-art P25 TiO2 nanoparticles (NPs). In addition, we have revealed the morphological effects of p-DCHD NCs, long-term stability, and the photocatalytic degradation mechanisms. These results will open a new pathway for the further development and understanding of conjugated polymer photocatalysts toward various potential applications.