Nucleic Acid Chemistry
Nucleic Acid Drugs
Drug Delivery System
Exosome
93
Scopus Publications
Scopus Publications
Detection of Epigenetically Important 5-Formylcytosine Modifications Using Novel Photoreactive Oligonucleotides Containing a Trioxsalen-Conjugated Guanosine Yu Mikame, Hiroaki Shirahama, Kinuka Doi, Nagisa Maekawa, Hiroki Kanazawa, Tsuyoshi Yamamoto, Chikara Dohno, Jiro Kondo, Takehiko Wada, Asako Yamayoshi Journal of the American Chemical Society, 2026 Transcription is tightly regulated by epigenetic modifications that control the expression of specific genes. One such modification is 5-methylcytosine (5mC) that is formed by cytosine methylation via DNA methyltransferases and functions as a transcriptional silencer of gene promoters. Dynamically regulated subsequent modifications by ten-eleven translocation (TET) enzymes catalyze the sequential oxidation of 5mC for generating 5-hydroxymethylcytosine, 5-formylcytosine (5fC), and 5-carboxylcytosine. Recently, these oxidized cytosine derivatives were indicated as not merely intermediates in the demethylation process; however, each derivative plays a unique biologically relevant role. A photo-crosslinkable oligonucleotide probe for detecting these cytosine modifications could support the spatiotemporal functional analysis of cytosine derivatives via a light stimulus. Herein, we designed novel photoreactive nucleosides by conjugating a psoralen (Ps) derivative, trioxsalen, to the C2' position of guanosine (GPs and GPs2) and developed photo-crosslinkable oligonucleotides (GPs-Oligo and GPs2-Oligo). In particular, GPs-Oligo demonstrated an intriguing and unique ability of photo-crosslinking with 5fC. GPs-Oligo was employed to detect 5fC using a DNA chip glass plate, which demonstrated the potential of GPs-Oligo for future investigation of the function of 5fC inside cells.
Development and functional evaluation of a psoralen-conjugated nucleoside mimic for triplex-forming oligonucleotides Yu Mikame, Haruki Toyama, Chikara Dohno, Takehiko Wada, Asako Yamayoshi Communications Chemistry, 2025 Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been employed for the photodynamic regulation of gene expression by the photo-cross-linking of psoralen with the target DNA. However, stable triplex formation requires a consecutive purine base sequence in one strand of the target DNA duplexes. The pyrimidine-base interruption in the consecutive purine base sequence drastically decreases the thermodynamic stability of the corresponding triplex, which hampers the TFO application. Here, we propose a design of the Ps-TFO for stable triplex formation with target DNA sequences containing pyrimidine-base interruptions under physiological conditions. This Ps-TFO, named 1’(one)-psoralen-conjugated triplex-forming oligonucleotide (OPTO), incorporates a synthesized nucleoside mimic 1’-psoralen-conjugated deoxyribose to increase the thermodynamic stability of the corresponding triplex by the intercalation of psoralen. The triplex-forming abilities of the OPTO were successfully demonstrated in combination with LNA and 5-methylcytosine, indicating that the use of OPTO will expand the range of the target sequences of TFO for photodynamic gene regulation. Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been employed for the photodynamic regulation of gene expression by the photo-cross-linking of psoralen with the target DNA, however, thermodynamic stability remains a limitation, hampering the application of TFO. Here, the authors design a 1’(one)-psoralen-conjugated triplex-forming oligonucleotide to achieve stable parallel triplex formation and photo-cross-linking formation with pyrimidine-base-interrupting sequences under physiological conditions.
Efficacy of DNA Intercalator-Conjugated Triplex-Forming Oligonucleotide as Anticancer Agent Haruki Toyama, Akira Toriba, Atsushi Shibata, Takehiko Wada, Asako Yamayoshi, Yu Mikame Chemmedchem, 2025 A triplex‐forming oligonucleotide (TFO) can form a sequence‐specific triple helix via Hoogsteen hydrogen bonding to polypurine tracts within a major groove side of a DNA duplex. Triplex formation can induce a double‐strand break, and this phenomenon at the amplified gene loci can selectively induce the cell death of cancer cells with specific gene amplification. However, the relationship between the binding affinity of TFO for target gene loci and the cell death response remains unclear. In this study, it is aimed to develop DNA intercalator‐conjugated TFOs with higher affinity for the human epidermal growth factor receptor type2 gene, which is often amplified in breast cancer cells, than the unmodified TFO. The binding affinity of the TFOs for the target DNA duplex is analyzed using nondenaturing polyacrylamide gel electrophoresis, and one of the DNA intercalator‐conjugated TFOs show a higher binding affinity for the target duplex than the unmodified TFO. The cell death responses induced by these TFOs using the WST‐8 assay is also evaluated suggesting that the higher binding affinity of the TFO for amplified gene loci can lead to a stronger cell death response of cancer cells with specific gene amplification.
Publisher Correction: Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry (Nature Communications, (2023), 14, 1, (7972), 10.1038/s41467-023-43714-0) Chisato Terada, Kaho Oh, Ryutaro Tsubaki, Bun Chan, Nozomi Aibara, Kaname Ohyama, Masa-Aki Shibata, Takehiko Wada, Mariko Harada-Shiba, Asako Yamayoshi, Tsuyoshi Yamamoto Nature Communications, 2024 Authors and Affiliations Department of Chemistry of Biofunctional Molecules, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan Chisato Terada, Kaho Oh, Ryutaro Tsubaki, Asako Yamayoshi & Tsuyoshi Yamamoto JSPS Research Fellow (DC1), Japan Society for the Promotion of Science, Tokyo, Japan Chisato Terada Graduate School of Engineering, Nagasaki University, Nagasaki, Japan Bun Chan Department of Pharmacy Practice, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan Nozomi Aibara Department of Molecular Pathochemistry, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan Kaname Ohyama Department of Anatomy and Cell Biology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Japan Masa-Aki Shibata Institute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku University, Sendai, Miyagi, Japan Takehiko Wada Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, Suita, Japan Mariko Harada-Shiba Cardiovascular Center, Osaka Medical and Pharmaceutical University, Takatsuki, Japan Mariko Harada-Shiba Authors Chisato Terada View author publications You can also search for this author in PubMed Google Scholar Kaho Oh View author publications You can also search for this author in PubMed Google Scholar Ryutaro Tsubaki View author publications You can also search for this author in PubMed Google Scholar Bun Chan View author publications You can also search for this author in PubMed Google Scholar Nozomi Aibara View author publications You can also search for this author in PubMed Google Scholar Kaname Ohyama View author publications You can also search for this author in PubMed Google Scholar Masa-Aki Shibata View author publications You can also search for this author in PubMed Google Scholar Takehiko Wada View author publications You can also search for this author in PubMed Google Scholar Mariko Harada-Shiba View author publications You can also search for this author in PubMed Google Scholar Asako Yamayoshi View author publications You can also search for this author in PubMed Google Scholar Tsuyoshi Yamamoto View author publications You can also search for this author in PubMed Google Scholar Corresponding author Correspondence to Tsuyoshi Yamamoto .
Modular Synthesis of Methyl-Substituted Novel Psoralen N -Hydroxysuccinimide Esters and Evaluation of DNA Photocrosslinking Properties of the Corresponding Triplex-Forming Oligonucleotide Conjugates Yu Mikame, Nagisa Maekawa, Soichiro Kimura, Juki Nakao, Asako Yamayoshi Synlett, 2024 Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been used to induce DNA mutations or suppress gene expression via the formation of crosslinked products with DNA in a sequence-specific manner. Psoralen can crosslink with DNA at its furan ring and/or pyrone ring side, yielding either a monoadduct or diadduct (interstrand crosslinking) product. The differences in the crosslinked structures of Ps-TFOs with the target DNAs are closely related to the changes in the biological outcomes induced by the Ps-TFOs. However, only few reports have discussed the photo-crosslinking properties of Ps-TFOs. The photo-crosslinking properties of Ps-TFOs with structurally diverse psoralen derivatives remain elusive. Herein, we report the modular synthesis of methyl-substituted novel psoralen N-hydroxysuccinimide (NHS) esters. Using these esters, the effect of the methyl substituent of psoralen on the photo-crosslinking of the corresponding Ps-TFOs was examined. Results revealed that the amount of the diadduct product was significantly reduced in the presence of the methyl substituents at C-3 and C-4 position while maintaining the total amount of photo-crosslinking product. This work demonstrates the possibility to control the crosslinked product of Ps-TFOs by introducing methyl groups into psoralen—this ability to manipulate the product is an important factor in the biological applications of Ps-TFOs.
Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry Chisato Terada, Kaho Oh, Ryutaro Tsubaki, Bun Chan, Nozomi Aibara, Kaname Ohyama, Masa-Aki Shibata, Takehiko Wada, Mariko Harada-Shiba, Asako Yamayoshi, Tsuyoshi Yamamoto Nature Communications, 2023 Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.
Development and Crosslinking Properties of Psoralen-Conjugated Triplex-Forming Oligonucleotides as Antigene Tools Targeting Genome DNA Yu Mikame, Honoka Eshima, Haruki Toyama, Juki Nakao, Misaki Matsuo, Tsuyoshi Yamamoto, Yoshiyuki Hari, Jun A. Komano, Asako Yamayoshi Chemmedchem, 2023 Psoralen‐conjugated triplex‐forming oligonucleotides (Ps‐TFOs) have been utilized for genome editing and anti‐gene experiments for over thirty years. However, the research on Ps‐TFOs employing artificial nucleotides is still limited, and their photo‐crosslinking properties have not been thoroughly investigated in relation to biological activities. In this study, we extensively examined the photo‐crosslinking properties of Ps‐TFOs to provide fundamental insights for future Ps‐TFO design. We developed novel Ps‐TFOs containing 2′‐O,4′‐C‐methylene‐bridged nucleic acids (Ps‐LNA‐mixmer) and investigated their photo‐crosslinking properties using stable cell lines that express firefly luciferase constitutively to evaluate the anti‐gene activities of Ps‐LNA‐mixmer. As a result, Ps‐LNA‐mixmer successfully demonstrated suppression activity, and we presented the first‐ever correlation between photo‐crosslinking properties and their activities. Our findings also indicate that the photo‐crosslinking process is insufficient under cell irradiation conditions (365 nm, 2 mW/cm2, 60 min). Therefore, our results highlight the need to develop new psoralen derivatives that are more reactive under cell irradiation conditions.
Exosome-Hijacking Drug Delivery System with Branched Arginine Linker Effectively Deliver Antisense Oligonucleotides into Lung Adenocarcinoma Cells Shota Oyama, Mao Tomita, Moeka Hata, Yu Mikame, Tsuyoshi Yamamoto, Eishi Ashihara, Asako Yamayoshi Chemical and Pharmaceutical Bulletin, 2023 Exosomes are a type of extracellular vesicles that contain diverse molecules and are present in our body fluids. They play a crucial role in transporting materials and transmitting signals between cells. Currently, there have been numerous reports on the use of exosomes in drug delivery systems (DDS). However, most existing methods for utilizing exosomes in DDS require the isolation and purification of exosomes, which raises concerns about yield and potential damage to the exosomes.Recently, we have developed a novel DDS called "ExomiR-Tracker" that harnesses exosomes without the need for isolation and purification. This system aims to deliver nucleic acid drugs effectively. ExomiR-Tracker consists of an anti-exosome antibody equipped with nona-D-arginines (9 mer) and nucleic acid drugs which have complementary sequence of target microRNA (anti-miR). In this study, we modified ExomiR-Tracker by incorporating branched nona-D-arginines (9 + 9 mer) molecules (referred to as Branch ExomiR-Tracker) and evaluated its efficacy in lung adenocarcinoma cells (A549 cells). The improved complex formation ability and enhanced cellular uptake of anti-miR, demonstrated by our findings, highlight the advantages of incorporating branched oligoarginine peptides into the ExomiR-Tracker platform. These results represent significant progress in revealing the effectiveness of Branch ExomiR-Tracker against adhesive cancer cells, which has not been shown to be effective with the conventional Linear ExomiR-Tracker.
Recent Advancements in Development and Therapeutic Applications of Genome-Targeting Triplex-Forming Oligonucleotides and Peptide Nucleic Acids Yu Mikame, Asako Yamayoshi Pharmaceutics, 2023 Recent developments in artificial nucleic acid and drug delivery systems present possibilities for the symbiotic engineering of therapeutic oligonucleotides, such as antisense oligonucleotides (ASOs) and small interfering ribonucleic acids (siRNAs). Employing these technologies, triplex-forming oligonucleotides (TFOs) or peptide nucleic acids (PNAs) can be applied to the development of symbiotic genome-targeting tools as well as a new class of oligonucleotide drugs, which offer conceptual advantages over antisense as the antigene target generally comprises two gene copies per cell rather than multiple copies of mRNA that are being continually transcribed. Further, genome editing by TFOs or PNAs induces permanent changes in the pathological genes, thus facilitating the complete cure of diseases. Nuclease-based gene-editing tools, such as zinc fingers, CRISPR-Cas9, and TALENs, are being explored for therapeutic applications, although their potential off-target, cytotoxic, and/or immunogenic effects may hinder their in vivo applications. Therefore, this review is aimed at describing the ongoing progress in TFO and PNA technologies, which can be symbiotic genome-targeting tools that will cause a near-future paradigm shift in drug development.
Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen N-Hydroxysuccinimide Esters Juki Nakao, Yu Mikame, Honoka Eshima, Tsuyoshi Yamamoto, Chikara Dohno, Takehiko Wada, Asako Yamayoshi Chembiochem, 2023 Psoralens and their derivatives, such as trioxsalen, have unique crosslinking features to DNA. However, psoralen monomers do not have sequence-specific crosslinking ability with the target DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable, thereby expanding the application of psoralen-conjugated molecules in gene transcription inhibition, gene knockout, and targeted recombination by genome editing. In this study, we developed two novel psoralen N-hydroxysuccinimide (NHS) esters that allow the introduction of psoralens into any amino-modified oligonucleotides. Quantitative evaluation of the photo-crosslinking efficiencies of the Ps-Oligos to target single-stranded DNAs revealed that the crosslinking selectivity to 5-mC is the unique feature of trioxsalen. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target double-stranded DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools.
