Faculty Profiles - ABE Hiroshi

Papers - ABE Hiroshi

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Synthesis of hydrophobic-tagged 2′-deoxy-modified cap analogs and its effect on mRNA translation

Zheyu Meng, Yuko Nakashima, Masahito Inagaki, Zhenmin Li, Susit Acharyya, Fumitaka Hashiya, Naoko Abe, Yasuaki Kimura, Hiroshi Abe

BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN Vol. 98 ( 2 ) 2025.2

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Messenger RNA (mRNA) has been implemented as COVID-19 vaccines. Following their success, the application of mRNA in cancer vaccines and protein replacement therapies is highly anticipated. The translational efficiency of mRNA largely depends on the cap structure located at its 5 ' end. In particular, the Cap-1 and Cap-2 structures are known to exhibit higher protein synthesis levels than the Cap-0 structure. Previously, we developed a method called the PureCap technique, which utilizes reversed-phase high-performance liquid chromatography (RP-HPLC) to purify highly capped mRNA with high precision, enabling accurate evaluation of translational activity. In this study, we synthesized cap analogs incorporating deoxyribose, and evaluated their mRNA synthesis efficiency and translational activity. Furthermore, by optimizing the transcription reaction conditions using the synthesized DNA-modified Cap series, we successfully improved the capping efficiency and mRNA yield. The deoxyadenosine-modified cap analog synthesized using the PureCap technique demonstrated 3.2-fold higher translational activity than the standard Cap-1 structure. Additionally, when using tetranucleotide cap analogs, consistent translational activity was observed, regardless of base sequence differences. This confirmed the general applicability of DNA-modified mRNA for translational activity.

DOI： 10.1093/bulcsj/uoaf006

Web of Science

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Internal cap-initiated translation for efficient protein production from circular mRNA

Kosuke Fukuchi, Yuko Nakashima, Naoko Abe, Seigo Kimura, Fumitaka Hashiya, Yuichi Shichino, Yiwei Liu, Ryoko Ogisu, Satomi Sugiyama, Daisuke Kawaguchi, Masahito Inagaki, Zheyu Meng, Shiryu Kajihara, Mizuki Tada, Satoshi Uchida, Ting-Ting Li, Ramkrishna Maity, Tairin Kawasaki, Yasuaki Kimura, Shintaro Iwasaki, Hiroshi Abe

NATURE BIOTECHNOLOGY 2025.2

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PORTFOLIO

<title>Abstract</title>
Circular mRNA, emerging as a groundbreaking RNA therapeutic strategy, faces challenges in enhancing its translation potential. Here, we introduce two innovative molecular designs that bolster circular mRNA translation through an internal cap-initiated mechanism. The first design involved a circular mRNA with a covalently attached <italic>N</italic>7-methylguanosine (m7G) cap through a branching structure (cap-circ mRNA). This modification allows circular mRNA to recruit translation machinery and produce proteins more efficiently than IRES-containing circular mRNAs. Combining <italic>N</italic>1-methylpseudouridine (m1Ψ) modification, cap-circ mRNA exhibits a lower acute immunostimulatory effect, maintaining high translation ability, in mice. The second design features the non-covalent attachment of an m7G cap to a circular mRNA through hybridization with an m7G cap-containing oligonucleotide, significantly enhancing translation by more than 50-fold. This setup allows the design of circular mRNAs to synthesize reporter proteins upon hybridizing with capped mRNAs or long non-coding RNAs and to undergo rolling circle-type translation. These advancements have broadened the therapeutic applications of circular mRNA by minimizing their molecular size, elevating translation efficiency, and facilitating cell-type selective translation.

DOI： 10.1038/s41587-025-02561-8

Covalently or non-covalently attached m7G cap enhances protein production from circular mRNA Invited Reviewed International journal

Hiroshi Abe, Naoko Abe

NATURE BIOTECHNOLOGY 2025.2

Concise Affinity-Based Purification of Ligated mRNA for Structure-Activity Relationship Studies of Nucleosugar Modification Patterns Reviewed International journal

Hiroki Yamada, Hiroto Iwai, Fumitaka Hashiya, Yasuaki Kimura, Hiroshi Abe, Junichiro Yamamoto

CHEMBIOCHEM Vol. 26 ( 2 ) page： e202400711 2025.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-V C H VERLAG GMBH

Position-specific nucleoside sugar modifications have been shown to improve the translational activity and stability of chemically synthesized mRNA. For pharmaceutical applications of chemically modified mRNA, a rapid purification methodology is imperative to identify the optimal modification pattern. However, while the chemical synthesis of mRNAs can be accomplished by splint ligation of oligonucleotide fragments, the current purification method for ligated mRNAs based on denaturing polyacrylamide gel electrophoresis tends to be time consuming. In this study, we developed a two-step affinity purification method for rapid sample preparation. In this method, ligated mRNA is captured by oligo dT magnetic beads and streptavidin magnetic beads with 3'-biotinylated oligo DNA, which are complementary to the 3'-poly(A) and 5' terminal sequences of the target mRNA, respectively. Therefore, the target mRNA can be isolated from a complex mixture of splint ligations. Using this method, six sugar-modified mRNAs were simultaneously purified, and the translational activities of these mRNAs were evaluated immediately after purification. The results demonstrate that this methodology is suitable for the rapid preparation of various chemically synthesized mRNAs to identify their optimal modification patterns.

DOI： 10.1002/cbic.202400711

Synthesis of Novel Cap Analogs Bearing Hydrophobic Purification Tag Toward Application for Preparation of Fully Capped Messenger RNA

Inagaki M., Abe H.

Yuki Gosei Kagaku Kyokaishi/Journal of Synthetic Organic Chemistry Vol. 83 ( 1 ) page： 2 - 14 2025.1

Publisher：Yuki Gosei Kagaku Kyokaishi/Journal of Synthetic Organic Chemistry

Messenger RNA (mRNA) is gaining attention as a new modality of oligonucleotide therapeutics. We have reported a new mRNA synthesis method, named the PureCap method, by in vitro transcription, which enables the production of completely capped mRNA. For this purpose, we designed and synthesized new cap analogs bearing hydrophobic o–nitrobenzyl groups, which function as purification tags for mRNA with a 5́–cap structure by reverse–phase high–performance liquid chromatography. In this paper, we describe the synthesis of new cap analogs, named PureCap analogs, and its application for completely capped mRNA synthesis.

DOI： 10.5059/yukigoseikyokaishi.83.2

Scopus

Synthesis of 2′-formamidonucleoside phosphoramidites for suppressing the seed-based off-target effects of siRNAs Reviewed International journal Open Access

Kohei Nomura, Seongjin An, Yoshiaki Kobayashi, Jiro Kondo, Ting Shi, Hirotaka Murase, Kosuke Nakamoto, Yasuaki Kimura, Naoko Abe, Kumiko Ui-Tei, Hiroshi Abe

Nucleic Acids Research Vol. 52 ( 18 ) page： 10754 - 10774 2024.9

Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

Abstract

In this study, we report the synthesis of 2′-formamidonucleoside phosphoramidite derivatives and their incorporation into siRNA strands to reduce seed-based off-target effects of small interfering RNAs (siRNAs). Formamido derivatives of all four nucleosides (A, G, C and U) were synthesized in 5–11 steps from commercial compounds. Introducing these derivatives into double-stranded RNA slightly reduced its thermodynamic stability, but X-ray crystallography and CD spectrum analysis confirmed that the RNA maintained its natural A-form structure. Although the introduction of the 2′-formamidonucleoside derivative at the 2nd position in the guide strand of the siRNA led to a slight decrease in the on-target RNAi activity, the siRNAs with different sequences incorporating 2′-formamidonucleoside with four kinds of nucleobases into any position other than 2nd position in the seed region revealed a significant suppression of off-target activity while maintaining on-target RNAi activity. This indicates that 2′-formamidonucleosides represent a promising approach for mitigating off-target effects in siRNA therapeutics.

DOI： 10.1093/nar/gkae741

2′-β-Methylselenyl nucleos(t)ide analogs as reverse transcriptase inhibitors against diverse HIV mutants Reviewed International journal Open Access

Yuki Yoshida, Yushi Niimi, Daichi Fushihara, Hideo Katakura, Ryusuke Fukui, Hirotaka Murase, Fumiaki Tomoike, Fumitaka Hashiya, Tsutomu Murakami, Eiichi N. Kodama, Tetsuro Suzuki, Kiyoshi Yasukawa, Yasuaki Kimura, Hiroshi Abe

Bioorganic & Medicinal Chemistry Vol. 110 page： 117813 - 117813 2024.8

Intracellular delivery of antisense oligonucleotides by tri-branched cyclic disulfide units. Reviewed International journal Open Access

Fangjie Lyu, Hayase Hakariya, Haruka Hiraoka, Zhenmin Li, Noriaki Matsubara, Yonghao Soo, Fumitaka Hashiya, Naoko Abe, Zhaoma Shu, Kosuke Nakamoto, Yasuaki Kimura, Hiroshi Abe

ChemMedChem Vol. 19 ( 20 ) page： e202400472 2024.7

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Therapeutic oligonucleotides, such as antisense DNA, show promise in treating previously untreatable diseases. However, their applications are still hindered by the poor membrane permeability of naked oligonucleotides. Therefore, it is necessary to develop efficient methods for intracellular oligonucleotide delivery. Previously, our group successfully developed disulfide-based Membrane Permeable Oligonucleotides (MPON), which achieved enhanced cellular uptake and gene silencing effects through an endocytosis-free uptake mechanism. Herein, we report a new molecular design for the next generation of MPON, called trimer MPON. The trimer MPON consists of a tri-branched backbone, three α-lipoic acid units, and a spacer linker between the oligonucleotides and tri-branched cyclic disulfide unit. We describe the design, synthesis, and functional evaluation of the trimer MPON, offering new insights into the molecular design for efficient oligonucleotide delivery.

DOI： 10.1002/cmdc.202400472

Development and Comparison of 4-Thiouridine to Cytidine Base Conversion Reaction Invited Reviewed International journal Open Access

Sana Ohashi, Mayu Nakamura, Susit Acharyya, Masahito Inagaki, Naoko Abe, Yasuaki Kimura, Fumitaka Hashiya, Hiroshi Abe

ACS Omegam Vol. 9 ( 8 ) page： 9300 - 9308 2024.2

Development of PCR Primer Enabling the Design of Flexible Sticky Ends for Efficient Concatenation of Long DNA Fragments Reviewed International journal Open Access

Kohei Nomura, Kaoru Onda, Hirotaka Murase, Fumitaka Hashiya, Yukiteru Ono, Goro Terai, Natsuhisa Oka, Kiyoshi Asai, Daisuke Suzuki, Naho Takahashi, Haruka Hiraoka, Masahito Inagaki, Yasuaki Kimura, Yoshihiro Shimizu, Naoko Abe, Hiroshi Abe

RSC Chemical Biology Vol. 5 ( 4 ) page： 360 - 371 2024.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry (RSC)

We developed chemically modified PCR primers that allow the design of flexible sticky ends by introducing a photo-cleavable group at the phosphate moiety. Nucleic acid derivatives containing o-nitrobenzyl photo-cleavable groups...

DOI： 10.1039/d3cb00212h

Topological capture of mRNA for silencing gene expression. Reviewed International journal

Fangjie Lyu, Takashi Tomita, Naoko Abe, Haruka Hiraoka, Fumitaka Hashiya, Yuko Nakashima, Shiryu Kajihara, Fumiaki Tomoike, Zhaoma Shu, Kazumitsu Onizuka, Yasuaki Kimura, Hiroshi Abe

Chemical communications (Cambridge, England) Vol. 59 ( 77 ) page： 11564 - 11567 2023.9

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ROYAL SOC CHEMISTRY

We describe herein topological mRNA capture using branched oligodeoxynucleotides (ODNs) with multiple reactive functional groups. These fragmented ODNs efficiently formed topological complexes on template mRNA in vitro. In cell-based experiments targeting AcGFP mRNA, the bifurcated reactive ODNs showed a much larger gene silencing effect than the corresponding natural antisense ODN.

DOI： 10.1039/d2cc06189a

The effect of γ phosphate modified deoxynucleotide substrates on PCR activity and fidelity. Reviewed International journal Open Access

Fumitaka Hashiya, Hirotaka Murase, Akash Chandela, Haruka Hiraoka, Masahito Inagaki, Yuko Nakashima, Naoko Abe, Mayu Nakamura, Goro Terai, Yasuaki Kimura, Kaori Ando, Natsuhisa Oka, Kiyoshi Asai, Hiroshi Abe

Chembiochem : a European journal of chemical biology Vol. 24 ( 14 ) page： e202200572 2023.5

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Controlling PCR fidelity is an important issue for molecular biology and high-fidelity PCR is essential for gene cloning. In general, fidelity control is achieved by protein engineering of polymerases. In contrast, only a few studies have reported controlling fidelity using chemically modified nucleotide substrates. In this report, we synthesized nucleotide substrates possessing a modification on Pγ and evaluated the effect of this modification on PCR fidelity. One of the substrates, nucleotide tetraphosphate, caused a modest decrease in Taq DNA polymerase activity and the effect on PCR fidelity was dependent on the type of mutation. The use of deoxyadenosine tetraphosphate enhanced the A:T→G:C mutation dramatically, which is common when using Taq polymerase. Conversely, deoxyguanosine tetraphosphate (dG4P) suppressed this mutation but increased the G:C→A:T mutation during PCR. Using an excess amount of dG4P suppressed both mutations successfully and total fidelity was improved.

DOI： 10.1002/cbic.202200572

Synthesis of nucleoside oligophosphates by electrophilic activation of phosphorothioate. Reviewed International journal

Shogo Hasegawa, Masahito Inagaki, Shunichi Kato, Zhenmin Li, Yasuaki Kimura, Hiroshi Abe

Organic & biomolecular chemistry Vol. 21 ( 19 ) page： 3997 - 4001 2023.5

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry (RSC)

We herein report a new synthetic method of nucleoside oligophosphates based on an electrophilic activation of 5'-phosphorothioate nucleotides. Treatment of the phosphorothioate with 2,4-dinitrochlorobenzene (DNCB) efficiently afforded the key activated...

DOI： 10.1039/d2ob02260e

Cap analogs with a hydrophobic photocleavable tag enable facile purification of fully capped mRNA with various cap structures. Reviewed International journal Open Access

Masahito Inagaki, Naoko Abe, Zhenmin Li, Yuko Nakashima, Susit Acharyya, Kazuya Ogawa, Daisuke Kawaguchi, Haruka Hiraoka, Ayaka Banno, Zheyu Meng, Mizuki Tada, Tatsuma Ishida, Pingxue Lyu, Kengo Kokubo, Hirotaka Murase, Fumitaka Hashiya, Yasuaki Kimura, Satoshi Uchida, Hiroshi Abe

Nature communications Vol. 14 ( 1 ) page： 2657 - 2657 2023.5

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society (ACS)

Removing immunogenic uncapped mRNA from in vitro transcribed mRNA is critical in mRNA research and clinical applications. Commonly used capping methods provide a maximum capping efficiency of around 80-90% for widely used Cap-0- and Cap-1-type mRNAs. However, uncapped and capped mRNA possesses almost identical physicochemical properties, posing challenges to their physical separation. Herein, we developed hydrophobic photocaged tag-modified cap analogs, which separated capped mRNA from uncapped mRNA by reversed-phase HPLC. Subsequent photo-irradiation recovers footprint-free native capped mRNA. This approach provided 100% capping efficiency even in Cap-2-type mRNA with versatility applicable to 650 nt and 4,247 nt mRNA. The Cap-2-type mRNA showed up to 3 to 4-fold higher translational activity in cultured cells and animals than mRNA prepared by the standard capping method. Notably, the purification process simultaneously removed immunogenic double-stranded mRNA, another major contaminant of in vitro transcribed mRNA, drastically reducing mRNA immunogenicity in cultured cells.

DOI： 10.1038/s41467-023-38244-8

Creation of single molecular conjugates of metal-organic cages and DNA. Reviewed International journal Open Access

Toshinobu Nakajo, Shinpei Kusaka, Haruka Hiraoka, Kohei Nomura, Noriaki Matsubara, Rintaro Baba, Yuki Yoshida, Kosuke Nakamoto, Masakazu Honma, Hiroaki Iguchi, Takayuki Uchihashi, Hiroshi Abe, Ryotaro Matsuda

Chemical communications (Cambridge, England) Vol. 59 ( 33 ) page： 4974 - 4977 2023.4

Language：English Publishing type：Research paper (scientific journal)

Here we report the development of an equimolar conjugate of a metal-organic cage (MOC) and DNA (MOC-DNA). Several MOC-DNA conjugates were assembled into a programmed structure by coordinating with a template DNA having a complementary base sequence. Moreover, conjugation with the MOC drastically enhanced the permeability of DNA through the lipid bilayer, presenting great potential as a drug delivery system.

DOI： 10.1039/d3cc00460k

Use of Iontophoresis Technology for Transdermal Delivery of a Minimal mRNA Vaccine as a Potential Melanoma Therapeutic. Reviewed Open Access

Rabab A Husseini, Naoko Abe, Tomoaki Hara, Hiroshi Abe, Kentaro Kogure

Biological & pharmaceutical bulletin Vol. 46 ( 2 ) page： 301 - 308 2023

Language：English Publishing type：Research paper (scientific journal)

mRNA vaccines have attracted considerable attention as a result of the 2019 coronavirus pandemic; however, challenges remain regarding use of mRNA vaccines, including insufficient delivery owing to the high molecular weights and high negative charges associated with mRNA. These characteristics of mRNA vaccines impair intracellular uptake and subsequent protein translation. In the current study, we prepared a minimal mRNA vaccine encoding a tumor associated antigen human gp10025-33 peptide (KVPRNQDWL), as a potential treatment for melanoma. Minimal mRNA vaccines have recently shown promise at improving the translational process, and can be prepared via a simple production method. Moreover, we previously reported the successful use of iontophoresis (IP) technology in the delivery of hydrophilic macromolecules into skin layers, as well as intracellular delivery of small interfering RNA (siRNA). We hypothesized that combining IP technology with a newly synthesized minimal mRNA vaccine can improve both transdermal and intracellular delivery of mRNA. Following IP-induced delivery of a mRNA vaccine, an immune response is elicited resulting in activation of skin resident immune cells. As expected, combining both technologies led to potent stimulation of the immune system, which was observed via potent tumor inhibition in mice bearing melanoma. Additionally, there was an elevation in mRNA expression levels of various cytokines, mainly interferon (IFN)-γ, as well as infiltration of cytotoxic CD8+ T cells in the tumor tissue, which are responsible for tumor clearance. This is the first report demonstrating the application of IP for delivery of a minimal mRNA vaccine as a potential melanoma therapeutic.

DOI： 10.1248/bpb.b22-00746

Chemical synthesis of super messenger RNA medicine Reviewed

Tada Mizuki, Naoko Abe, Hiroshi Abe

Drug Delivery System Vol. 38 ( 1 ) page： 15 - 23 2023

Chemical synthesis of super messenger RNA medicine Reviewed Open Access

Tada M., Abe N., Abe H.

Drug Delivery System Vol. 38 ( 1 ) page： 15 - 23 2023

Authorship：Last author,　Corresponding author Language：Japanese Publishing type：Research paper (conference, symposium, etc.) Publisher：Drug Delivery System

Messenger RNA（mRNA）medicine was urgently approved in 2020 as a vaccine for COVID-19 . However, current mRNA therapeutics are not fully established, with challenges remaining in translation efficiency and drug delivery system. Therefore, further research is needed to adapt mRNA therapeutics to other diseases. Furthermore, the preparation of mRNA drugs is time-consuming and costly because of the biological methods used. Our laboratory has been working on chemical methods to solve these issues. In this paper, we introduce chemical modifications and novel capping reactions as a method to improve the translation efficiency of mRNA and the introduction of disulfide modification to oligonucleotide therapeutics as an effort on the drug delivery system.

DOI： 10.2745/dds.38.15

Open Access

Scopus

Complete chemical synthesis of mRNA

Kazuya Ogawa, Hiroshi Abe

2022.10

A 2'-modified uridine analog, 2'-O-(methylthiomethoxy)methyl uridine for siRNA applications Reviewed International journal Open Access

Lyu Fangjie, Seongjin An, Yoshiaki Kobayashi, Kohei Nomura, Rintaro Baba, Naoko Abe, Haruka Hiraoka, Fumitaka Hashiya, Zhaoma Shu, Kumiko Ui-Tei, Yasuaki Kimura, Hiroshi Abe

Bioorganic & Medicinal Chemistry Letters Vol. 74 page： 128939 - 128939 2022.8

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier BV

The medicinal applications of siRNAs have been intensively examined but are still hindered by their low molecular stability under biological conditions and off-target effects, etc. The introduction of chemical modifications to the nucleoside is a promising strategy for solving these limitations. Herein, we describe the development of a new uridine analog, U*, that has a (methylthiomethoxy)methoxy group at the 2' position. The phosphoramidite reagent corresponding to U* was easily synthesized and the RNA oligonucleotides containing U* were stably prepared using a standard protocol for oligonucleotide synthesis. The introduction of U* into the siRNA resulted in positive or negative effects on the targeted gene silencing in a position-dependent manner, and the positive effects were attributed to the improved stability under biological conditions. The thermodynamic analysis of the U*-modified RNAs revealed a slight destabilization of the dsRNA, based depending on which U was strategically utilized to restrain the off-target effects of the siRNA. This study describes a rare example of nucleoside analogs with a large substitution at the 2'-position in the context of an siRNA application and is informative for the development of other analogs to further improve the molecular properties of siRNAs for medicinal applications.

DOI： 10.1016/j.bmcl.2022.128939

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Papers - ABE Hiroshi