Oxidation-responsive supramolecular hydrogels based on glucosamine derivatives with an aryl sulfide group.

Supramolecular hydrogels can be obtained via self-assembly of small molecules in aqueous environments. In this study, we describe the development of oxidation-responsive supramolecular hydrogels comprising glucosamine derivatives with an aryl sulfide group. We demonstrate that hydrogen peroxide can induce a gel-sol transition through the oxidation of the sulfide group to the corresponding sulfoxide. Furthermore, we show that this oxidation responsiveness can be extended to photo-responsiveness with the aid of a photosensitizer.

Design of supramolecular hybrid nanomaterials comprising peptide-based supramolecular nanofibers and in situ generated DNA nanoflowers through rolling circle amplification.

The artificial construction of multicomponent supramolecular materials comprising plural supramolecular architectures that are assembled orthogonally from their constituent molecules has attracted growing attention. Here, we describe the design and development of multicomponent supramolecular materials by combining peptide-based self-assembled fibrous nanostructures with globular DNA nanoflowers constructed by the rolling circle amplification reaction. The orthogonally constructed architectures were dissected by fluorescence imaging using the selective fluorescence staining procedures adapted to this study. The present, unique hybrid materials developed by taking advantage of each supramolecular architecture based on their peptide and DNA functions may offer distinct opportunities to explore their bioapplications as a soft matrix.

Construction of a Reduction-responsive DNA Microsphere using a Reduction-cleavable Spacer based on a Nitrobenzene Scaffold.

Here, we describe the design and synthesis of a new reduction-cleavable spacer (RCS) based on a nitrobenzene scaffold for constructing reduction-responsive oligonucleotides according to standard phosphoramidite chemistry. In addition, we demonstrate that the introduction of the RCS in the middle of an oligonucleotide (30 nt) enables the construction of a self-assembled microsphere capable of exhibiting a reduction-responsive disassembly.

Formation of Supramolecular Nanostructures through in Situ Self-Assembly and Post-Assembly Modification of a Biocatalytically Constructed Dipeptide Hydrazide.

Aqueous self-assembly of short peptides has attracted growing attention for the construction of supramolecular materials for various bioapplications. Herein, we describe how the thermolysin-assisted biocatalytic construction of a dipeptide hydrazide from an N-protected amino acid and an amino acid hydrazide leads to the formation of thermally stable supramolecular hydrogels. In addition, we demonstrate the post-assembly modification of the supramolecular architectures constructed in situ tethering hydrazide groups as a chemical handle by means of fluorescence imaging.

Sulfonamide antibiotics inhibit RNAi by binding to human Argonaute protein 2 PAZ.

We found that sulfisomidine, a sulfonamide antibiotic, potently binds to the Piwi/Argonaute/Zwille (PAZ) domain of human Argonaute protein 2 and inhibits RNA interference (RNAi). To elucidate the effect on RNAi of strong affinity of the 3'-ends in small interfering RNA (siRNA) to the PAZ domain, chemically modified siRNAs bearing sulfisomidine at the 3'-end were synthesized.

Chemical stimulus-responsive supramolecular hydrogel formation and shrinkage of a hydrazone-containing short peptide derivative.

Artificial supramolecular nanostructures showing transient properties have attracted significant attention in recent years. New discoveries in this area may provide insights into a better understanding of the sophisticated organization of complex biomolecular systems. Nevertheless, research concerning such materials is still limited. Better knowledge of the chemical reactivity and corresponding molecular transformations of self-assembling molecules, which guide their assembly/disassembly, may provide an opportunity to construct transient supramolecular nanostructures capable of showing chemical stimulus responsiveness. Herein, we report a short peptide derivative containing a hydrazone bond, which shows transient hydrogel formation (no only sol-to-gel but also gel-to-shrunken gel phase transition) accompanied by continuous transformation and growth of supramolecular nanostructures triggered by hydrazone-oxime exchange reaction in response to hydroxylamine. Such controlled shrinkage behavior of supramolecular hydrogels in response to specific chemical stimuli has rarely been explored compared with conventional polymer hydrogel systems.

Hybrid Soft Nanomaterials Composed of DNA Microspheres and Supramolecular Nanostructures of Semi-artificial Glycopeptides.

Aqueous hybrid soft nanomaterials consisting of plural supramolecular architectures with a high degree of segregation (orthogonal coexistence) and precise hierarchy at the nano- and microscales, which are reminiscent of complex biomolecular systems, have attracted increasing attention. Remarkable progress has been witnessed in the construction of DNA nanostructures obtained by rational sequence design and supramolecular nanostructures of peptide derivatives through self-assembly under aqueous conditions. However, orthogonal self-assembly of DNA nanostructures and supramolecular nanostructures of peptide derivatives in a single medium has not yet been explored in detail. In this study, DNA microspheres, which can be obtained from three single-stranded DNAs, and three different supramolecular nanostructures (helical nanofibers, straight nanoribbons, and flowerlike microaggregates) of semi-artificial glycopeptides were simultaneously constructed in a single medium by a simple thermal annealing process, which gives rise to hybrid soft nanomaterials. Fluorescence imaging with selective staining of each supramolecular nanostructure uncovered the orthogonal coexistence of these structures with only marginal impact on their morphology. Additionally, the biostimuli-responsive degradation propensity of each supramolecular architecture is retained, and this may allow the construction of active soft nanomaterials exhibiting intelligent biofunctions.

A stimuli-responsive DNAzyme displaying Boolean logic-gate responses.

Introducing a desired stimuli-responsive function into catalytically active biomacromolecules is potentially useful in developing molecular tools for various bio-applications. In this paper, we discuss the development of a stimuli-responsive DNAzyme (catalytic deoxyribozyme) capable of displaying Boolean logic-gate responses.

Reduction-Responsive DNA Duplex Containing O 6-Nitrobenzyl-Guanine.

Stimuli-controlled structural transitions of nucleic acids have received growing attentions owing to their potential applications in the fields of chemical and synthetic biology. Here, we describe the development of reduction-responsive deoxyribonucleic acid (DNA) duplexes, in which guanine rings bearing a reduction-responsive cleavable nitrobenzyl (NB) group at the O 6 position (GNB) are introduced at defined positions. We demonstrate that the artificial NB group can be removed in response to reduction stimulus without the dissociation of the intermolecular duplex structure, which comprises a G-quadruplex forming nucleic acid strand with one GNB and its complementary sequence with one mismatch pair. Meanwhile, another duplex that comprised a G-quadruplex forming nucleic acid strand with two GNB and its complementary sequence with three mismatch pairs exhibited reduction-responsive structural transitions from intermolecular duplex to intramolecular quadruplex. These findings might be useful for the development of DNA architectures endowed with reduction-responsive functions.

[Development of a Fluorescence Probe for Live Cell Imaging].

Probes that detect specific biological materials are indispensable tools for deepening our understanding of various cellular phenomena. In live cell imaging, the probe must emit fluorescence only when a specific substance is detected. In this paper, we introduce a new probe we developed for live cell imaging. Glutathione S-transferase (GST) activity is higher in tumor cells than in normal cells and is involved in the development of resistance to various anticancer drugs. We previously reported the development of a general strategy for the synthesis of probes for detection of GST enzymes, including fluorogenic, bioluminogenic, and 19F-NMR probes. Arylsulfonyl groups were used as caging groups during probe design. The fluorogenic probes were successfully used to quantitate very low levels of GST activity in cell extracts and were also successfully applied to the imaging of microsomal MGST1 activity in living cells. The bioluminogenic and 19F-NMR probes were able to detect GST activity in Escherichia coli cells. Oligonucleotide-templated reactions are powerful tools for nucleic acid sensing. This strategy exploits the target strand as a template for two functionalized probes and provides a simple molecular mechanism for multiple turnover reactions. We developed a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe completed its reaction within 30 s of initiation and amplified the fluorescence signal from 0.5 pM target oligonucleotide by 1500 fold under isothermal conditions. Additionally, we applied the oligonucleotide-templated reaction for molecular releasing and peptide detection.

Introduction of 2-O-benzyl abasic nucleosides to the 3'-overhang regions of siRNAs greatly improves nuclease resistance.

Chemically modified siRNAs containing 2-O-benzyl-1-deoxy-d-ribofuranose (RHOBn) in their 3'-overhang region were significantly more resistant towards serum nucleases than siRNAs possessing the natural nucleoside in this region. The knockdown efficacies and binding affinities of these modified siRNAs to the recombinant human Argonaute protein 2 (hAgo2) PAZ domain were comparable with that of siRNA with a thymidine dimer at the 3'-end.

Nucleobase azide-ethynylribose click chemistry contributes to stabilizing oligonucleotide duplexes and stem-loop structures.

The formation of 1,4-disubstituted 1,2,3-triazoles through copper-catalyzed azide-alkyne cycloaddition (CuAAC) in oligonucleotides bearing 1-deoxy-1-ethynyl-ß-d-ribofuranose (RE) can have a positive impact on the stability of oligonucleotide duplexes and stem-loop structures.

Reduction-Responsive Guanine Incorporated into G-Quadruplex-Forming DNA.

Stimulus-responsive biomolecules are attractive targets to understand biomolecule behaviour as well as to explore their therapeutic and diagnostic applications. We demonstrate that a reduction-responsive cleavable group (chemically caged unit) introduced into the guanine ring enables modulation of the secondary structure transition of an oligonucleotide in a reduction-responsive and traceless manner leaving the unmodified oligonucleotide of interest. This simple but robust strategy could yield a variety of stimuli-responsive oligonucleotides.

Practical and Reliable Synthesis of 1,2-Dideoxy-d-ribofuranose and its Application in RNAi Studies.

We developed a practical and reliable method for synthesizing an abasic deoxyribonucleoside, 1,2-dideoxy-d-ribofuranose (dR(H)) via elimination of nucleobase from thymidine. To synthesize oligonucleotides bearing dR(H) by the standard phosphoramidite solid-phase method, dR(H) was converted to the corresponding phosphoramidite derivative and linked to a solid support (controlled pore glass resin). Chemically modified small interfering RNAs (siRNAs) possessing dR(H) at their 3'-overhang regions were synthesized. Introducing dR(H) to the 3'-end of the antisense strand of siRNA reduced its knockdown effect.

Rolling Circle Translation of Circular RNA in Living Human Cells.

We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought.

Fluorogenic probes using 4-substituted-2-nitrobenzenesulfonyl derivatives as caging groups for the analysis of human glutathione transferase catalyzed reactions.

We have synthesized a series of 4-substituted-2-nitrobenzene-sulfonyl compounds for caged fluorogenic probes and conducted a Hammett plot analysis using the steady-state kinetic parameters. The results revealed that the glutathione transferase (GST) alpha catalyzed reaction was dependent on the σ value in the same way as the non-enzymatic reaction, whereas the dependence of the σ value of the GST mu and pi was not as pronounced as that of GST alpha.

Very rapid DNA-templated reaction for efficient signal amplification and its steady-state kinetic analysis of the turnover cycle.

Oligonucleotide-templated reactions are powerful tools for the detection of nucleic acid sequences. One of the major scientific challenges associated with this technique is the rational design of non-enzyme-mediated catalytic templated reactions capable of multiple turnovers that provide high levels of signal amplification. Herein, we report the development of a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe underwent a rapid templated reaction without any of the undesired background reactions. The fluorogenic reaction conducted in the presence of a template provided a 223-fold increase in fluorescence after 30 s compared with the nontemplated reaction. The probe provided an efficient level of signal amplification that ultimately enabled particularly sensitive levels of detection. Assuming a simple model for the templated reactions, it was possible to estimate the rate constants of the chemical reaction in the presence and in the absence of the template. From these kinetic analyses, it was possible to confirm that an efficient turnover cycle had been achieved, on the basis of the dramatic enhancement in the rate of the chemical reaction considered to be the rate-determining step. With maximized turnover efficiency, it was demonstrated that the probe could offer a high turnover number of 1500 times to enable sensitive levels of detection with a detection limit of 0.5 pM in the catalytic templated reactions.

RNA-templated molecule release induced protein expression in bacterial cells.

We have developed a system to release a biologically active molecule in response to the sequence of a target gene. The releasing system, which was triggered by the reduction of an azidomethyl group, was successfully applied to protein expression induced by the release of IPTG triggered by endogenous RNA in bacterial cells.

Structure formation and catalytic activity of DNA dissolved in organic solvents.

Equal-opportunity dissolver: By attaching polyethylene glycol at its 5' end, DNA (PEG-DNA) can be solubilized in various organic solvents and was shown to form G-quadruplexes by CD spectroscopy. A complex containing iron(III) protoporphyrin IX (hemin) and G-quadruplex-forming PEG-DNA catalyzed an oxidative reaction in methanol (see scheme).