Language：English   Publisher：Nature Communications  

Despite the remarkable success of mRNA vaccines, improving the translational efficiency of mRNA therapeutics remains a critical challenge to their widespread clinical application. Here we systematically evaluate chemical modifications to improve the translational activity and stability of uncapped mRNA. We employ a primarily chemistry-based synthetic approach, which is crucial for the position-specific introduction of chemical modifications, enabling detailed structure-activity relationship studies, hitherto unattainable with conventional methods. A pivotal innovation herein is the introduction of 2´-F modification at the first nucleoside of the codon in the open reading frame, which significantly bolsters the stability of mRNA without compromising its translation. Additional modifications at the 5´-UTR and poly(A) tail with other types of nucleoside and phosphate analogs also exemplify the importance of terminal modifications for improved translation. Precise control of these modification patterns achieves higher peptide expression than conventional in vitro-transcribed mRNA. These findings offer a unique framework for designing effective mRNA-based therapeutics.

DOI： 10.1038/s41467-025-65788-8

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Language：English   Publisher：Rsc Medicinal Chemistry  

Current LNP technology faces challenges that must be addressed to enhance the functionality of mRNA therapeutics. Recent studies show disulfide-conjugated molecules improve cell membrane permeability. Here, we investigated incorporating cyclic disulfide (CDL) units into lipid components of LNPs to enhance LNP-mRNA performance. A lipid library with branched and unbranched alkyl chains (C16-C20) and tertiary amine groups modified with CDLs was designed. While cellular uptake was unchanged, some mRNA-loaded LNPs with CDLs achieved more than 2-fold higher transfection efficiency than LNPs with MC3 or SM102 alone. Intracellular analysis revealed that the addition of CDL lipids significantly promoted endosomal escape. The CDL-incorporated LNPs administered subcutaneously in mice showed significantly higher luciferase gene expression compared to LNPs without CDL. Additionally, LNPs encapsulating OVA antigen-encoding mRNA induced a potent antitumor response against the EG7-OVA lymphoma model. These results suggest CDL modifications enhance LNP-based mRNA delivery, offering potential for broader therapeutic applications and improved clinical outcomes.

DOI： 10.1039/d5md00084j

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Language：English   Publisher：Vaccine  

Retinochoroidal neovascularization (NV), involved in macular degeneration, diabetic retinopathy, and other ocular diseases, causes vision impairment and blindness. Current treatments rely on repeated intraocular injections of anti-angiogenic drugs, which are burdensome for patients and clinicians, and some patients fail to respond to the treatments. This study investigates the potential of mRNA vaccination to mitigate NV and treat ocular pathologies. The vaccine targets leucine-rich alpha-2-glycoprotein 1 (LRG1), a protein specifically expressed in pathological neovascularization, inducing anti-LRG1 antibody responses in mice. In a laser-induced NV model, the LRG1 mRNA vaccine reduces NV area and leakage while inhibiting microglial cell infiltration. Histological analysis shows no adverse effects on retinal architecture or glial cell activation. Additionally, in Vldlr knockout mice, LRG1 mRNA administration suppresses ongoing neovascularization and downregulates key angiogenic mediators. These findings highlight the therapeutic potential of LRG1 mRNA as a novel strategy for CNV-associated diseases.

DOI： 10.1016/j.vaccine.2025.127451

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Language：English   Publisher：Communications Chemistry  

Chemical ligation of oligonucleotides enables assembly of long DNA constructs essential for genome engineering, DNA nanotechnology, and molecular diagnostics, but current methods often require external activators and suffer from reactive intermediate instability. Here we show a reagent-free DNA autoligation strategy based on intramolecular cross-activation between 3′-phosphorothioate (PS) and 5′-dinitrobenzenesulfonamide (DNBSA) termini on a splint DNA, yielding a P3′ → N5′ phosphoramidate linkage under near-physiological conditions. Ligation proceeds with over 80% yield at 37 °C and pH 8 without external reagents. The DNBSA group exhibits exceptional aqueous stability, and in situ formation of reactive intermediates contributes to high efficiency. This strategy expands the current toolkit for assembling DNA constructs and may facilitate future biotechnological and therapeutic studies.

DOI： 10.1038/s42004-025-01631-x

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Language：English   Publisher：Journal of Controlled Release  

Developing effective vaccines against bacteria is critical given the growing threat of antimicrobial resistance (AMR). In this study, we developed mRNA vaccines targeting Pseudomonas aeruginosa (P. aeruginosa), a key AMR pathogen, using PureCap mRNA encapsulated in lipid nanoparticles (LNPs). The PureCap technology offers a facile method for removing immunostimulatory impurities from in vitro transcribed mRNA, such as uncapped RNA and double-stranded RNA (dsRNA). Following intramuscular vaccination of mice with mRNA encoding a model antigen, PureCap mRNA elicited antibody titers that were 26-fold higher than those induced by conventional ARCA-capped mRNA. Mechanistic analyses revealed that both uncapped RNA and dsRNA impurities in ARCA-capped mRNA were responsible for the reduced humoral immune responses. While PureCap mRNA enhanced protein expression efficiency and reduced pro-inflammatory responses compared to ARCA-capped mRNA, minimizing pro-inflammatory responses was particularly critical. When anti-interferon-α/β receptor antibodies were administered, antibody responses to ARCA-capped mRNA vaccination were restored to levels comparable to those achieved with PureCap mRNA vaccination, highlighting the negative impact of type I interferon responses on antibody responses following vaccination with ARCA-capped mRNA. In a vaccination targeting the PcrV protein of P. aeruginosa, PureCap mRNA, but not ARCA-capped mRNA, significantly prolonged the survival of mice following bacterial challenges, presumably due to enhanced antibody production. Furthermore, PureCap mRNA vaccination significantly reduced bacterial loads in the lungs and mitigated tissue damage, edema, and inflammatory responses. These findings underscore the potential of PureCap mRNA as a promising platform for bacterial vaccination, offering a valuable strategy to combat AMR.

DOI： 10.1016/j.jconrel.2025.113860

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Language：English   Publisher：Briefings in Bioinformatics  

The computational design of messenger RNA (mRNA) sequences is a critical technology for both scientific research and industrial applications. Recent advances in prediction and optimization models have enabled the automatic scoring and optimization of 5' UTR sequences, key upstream elements of mRNA. However, fully automated design of 5' UTR sequences with more than two objective scores has not yet been explored. In this study, we present a computational pipeline that optimizes human 5' UTR sequences in a multi-objective framework, addressing up to four distinct and conflicting objectives. Our work represents an important advancement in the multi-objective computational design of mRNA sequences, paving the way for more sophisticated mRNA engineering.

DOI： 10.1093/bib/bbaf225

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Language：English   Publisher：The Pharmaceutical Society of Japan  

<p>The advent of mRNA medicine, initially implemented as a vaccine during the coronavirus disease 2019 (COVID-19) pandemic, has attracted interest in diverse therapeutic applications, including cancer vaccines and protein replacement therapies. Our group recently established a method for the complete chemical synthesis of mRNA. Although this approach has some advantages, chemically synthesized mRNA is limited to approximately 150 nucleotides in length and necessitates optimized designs for untranslated regions (UTRs) and coding sequences. To address this challenge, we investigated whether the non-reporter-based selection methods, including ribosome profiling and polysome profiling, which were often used for UTR optimization in long mRNA, could be adapted for short mRNA to identify highly translated UTR sequences. Using these methods, we collected mRNAs that interacted with ribosomes and analyzed their 5′-UTR sequences. We successfully identified a 9-nucleotide 5′-UTR that demonstrated approximately double the translation efficiency of the Kozak sequence, a widely used positive control. This work highlights the adaptability of ribosome-focused selection techniques for short, chemically synthesized mRNA and provides a foundation for effective sequence design. These findings advance the development of chemically synthesized mRNA as a viable alternative to <i>in vitro</i>-transcribed mRNA, paving the way for innovative therapeutic applications.</p>

DOI： 10.1248/cpb.c25-00048

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Publisher：Bulletin of the Chemical Society of Japan  

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

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Language：English   Publisher：Nucleic Acids Research  

We developed phosphorylation reagents with a nitrobenzyl hydrophobic tag and used them for 5′-phosphorylation of chemically or transcriptionally synthesized RNA. The capability of hydrophobic tags to synthesize 5′-monophosphorylated RNA was evaluated based on the yield of the desired oligonucleotides, stability of protecting groups during cleavage/deprotection, separation ability in reverse-phase HPLC (RP-HPLC), and deprotection efficiency after RP-HPLC purification. The results showed that a nitrobenzyl derivative with a tert-butyl group at the benzyl position was most suitable for RNA 5′-phosphorylation. Using the developed phosphorylation reagent, we chemically synthesized 5′-phosphorylated RNA and confirmed that it could be purified by RP-HPLC and the following deprotection. In addition, we demonstrated complete chemical synthesis of minimal mRNA by chemical capping of 5′-monophosphorylated RNA. Ribonucleoside 5′-monophosphates with hydrophobic protecting groups have also been developed and used as substrates to transcriptionally synthesize 5′-phosphorylated RNA with >1000 bases. From the mixture of the by-products and the desired RNA, only 5′-monophosphorylated RNA could be effectively isolated by RP-HPLC. Furthermore, monophosphorylated RNA can be converted into circular mRNA via RNA ligase-mediated cyclization. Circular mRNA expression of nanoluciferase in cultured cells and mice. These techniques are important for the production of chemically synthesized mRNA and circular mRNA.

DOI： 10.1093/nar/gkae847

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Language：English   Publisher：Chemmedchem  

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

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Language：English   Publisher：Nucleic Acids Research  

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

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Language：English   Publisher：Rsc Chemical Biology  

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 with a tert-butyl group at the benzyl position were stable during strong base treatment for oligonucleotide synthesis and thermal cycling in PCR reactions. PCR using primers incorporating these nucleic acid derivatives confirmed that chain extension reactions completely stopped at position 1 before and after the site of the photo-cleavable group was introduced. DNA fragments of 2 and 3 kbp, with sticky ends of 50 bases, were successfully concatenated with a high yield of 77%. A plasmid was constructed using this method. Finally, we applied this approach to construct a 48.5 kbp lambda phage DNA, which is difficult to achieve using restriction enzyme-based methods. After 7 days, we were able to confirm the generation of DNA of the desired length. Although the efficiency is yet to be improved, the chemically modified PCR primer offers potential to complement enzymatic methods and serve as a DNA concatenation technique.

DOI： 10.1039/d3cb00212h

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Language：English   Publisher：ACS Omega  

To study transcriptome dynamics without harming cells, it is essential to convert chemical bases. 4-Thiouridine (4sU) is a biocompatible uridine analogue that can be converted into a cytidine analogue. Although several reactions can convert 4sU into a cytidine analogue, few studies have compared the features of these reactions. In this study, we performed three reported base conversion reactions, including osmium tetroxide, iodoacetamide, and sodium periodate treatment, as well as a new reaction using 2,4-dinitrofluorobenzene. We compared the reaction time, conversion efficacy, and effects on reverse transcription. These reactions successfully converted 4sU into a cytidine analogue quantitatively using trinucleotides. However, the conversion efficacy and effect on reverse transcription vary depending on the reaction with the RNA transcript. OsO<inf>4</inf> treatment followed by NH<inf>4</inf>Cl treatment showed the best base-conversion efficiency. Nevertheless, each reaction has its own advantages and disadvantages as a tool for studying the transcriptome. Therefore, it is crucial to select the appropriate reaction for the target of interest.

DOI： 10.1021/acsomega.3c08516

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Language：English   Publisher：Chemical Communications  

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

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Language：English   Publisher：Chembiochem  

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 P<inf>γ</inf> 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

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Language：English   Publisher：Nature Communications  

Starting with the clinical application of two vaccines in 2020, mRNA therapeutics are currently being investigated for a variety of applications. Removing immunogenic uncapped mRNA from transcribed mRNA is critical in mRNA research and clinical applications. Commonly used capping methods provide 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. In this work, we develop hydrophobic photocaged tag-modified cap analogs, which separate capped mRNA from uncapped mRNA by reversed-phase high-performance liquid chromatography. Subsequent photo-irradiation recovers footprint-free native capped mRNA. This approach provides 100% capping efficiency even in Cap-2-type mRNA with versatility applicable to 650 nt and 4,247 nt mRNA. We find that the Cap-2-type mRNA shows up to 3- to 4-fold higher translation activity in cultured cells and animals than the Cap-1-type mRNA prepared by the standard capping method.

DOI： 10.1038/s41467-023-38244-8

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Language：Japanese   Publisher：The Pharmaceutical Society of Japan  

Recently, mRNA vaccines have been used with great success in SARS-CoV2 infections. However, mRNA vaccines are subject to reduced activity due to the <i>in vivo</i> instability of mRNA and other factors. Therefore, it is effective to introduce chemical modifications to mRNA that are expected to improve its stability, but there is no general synthetic method to achieve this. In this study, we developed a method for complete chemical synthesis of mRNA that avoids the enzymatic method, which is a barrier to the introduction of chemical modifications to mRNA. This method enables the introduction of site-specific chemical modifications to mRNA, and the appropriate chemical modification pattern that maximizes translational activity and stability. This paper describes the development of the complete chemical synthesis method, the structure-activity relationship of chemically modified mRNAs by the complete chemical synthesis method, and the therapeutic effects of mRNA cancer vaccines synthesized by this method.

DOI： 10.14894/medchem.33.2_75

CiNii Research

Language：English   Publisher：The Pharmaceutical Society of Japan  

<p>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 gp100_25–33 peptide (KVPRNQDWL), as a potential treatment for melanoma. Minimal mRNA vaccines have recently shown promise at improving the translational process, and can be prepared <i>via</i> 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 <i>via</i> 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.</p>

DOI： 10.1248/bpb.b22-00746

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CiNii Research

Language：Japanese   Publisher：THE JAPAN SOCIETY OF 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

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Language：English   Publisher：Bioorganic and Medicinal Chemistry Letters  

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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Language：English   Publisher：ACS Chemical Biology  

Site-specific chemical modification of mRNA can improve its translational efficiency and stability. For this purpose, it is desirable to develop a complete chemical synthesis method for chemically modified mRNA. The key is a chemical reaction that introduces a cap structure into the chemically synthesized RNA. In this study, we developed a fast and quantitative chemical capping reaction between 5′-phosphorylated RNA and N⁷-methylated GDP imidazolide in the presence of 1-methylimidazole in the organic solvent dimethyl sulfoxide. It enabled quantitative preparation of capping RNA within 3 h. We prepared chemically modified 107-nucleotide mRNAs, including N⁶-methyladenosine, insertion of non-nucleotide linkers, and 2′-O-methylated nucleotides at the 5′ end and evaluated their effects on translational activity in cultured HeLa cells. The results showed that mRNAs with non-nucleotide linkers in the untranslated regions were sufficiently tolerant to translation and that mRNAs with the Cap_2 structure had higher translational activity than those with the Cap_0 structure.

DOI： 10.1021/acschembio.1c00996

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Language：English   Publisher：Chembiochem  

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.

DOI： 10.1002/cbic.202100413

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Language：English   Publisher：Chembiochem  

Chemical ligation reaction of DNA is useful for the construction of long functional DNA using oligonucleotide fragments that are prepared by solid phase chemical synthesis. However, the unnatural linkage structure formed by the ligation reaction generally impairs the biological function of the resulting ligated DNA. We achieved the complete chemical synthesis of 78 and 258 bp synthetic DNAs via multiple chemical ligation reactions with phosphorothioate and haloacyl-modified DNA fragments. The latter synthetic DNA, coding shRNA for luciferase genes with a designed truncated SV promoter sequence, successfully induced the expected gene silencing effect in HeLa cells.

DOI： 10.1002/cbic.202100312

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Language：Japanese   Publisher：The Pharmaceutical Society of Japan  

Currently, mRNA is used for vaccine therapy against COVID-19. However, the mRNA vaccine is urgently approved under a pandemic, there are still issues to be solved in terms of delivery method, quality/ purity, stability/ effect sustainability, protein synthesis efficiency, and so on. Research and development of mRNA drugs have been mainly focused on the development of delivery methods, and only biological methods are used for mRNA production. It is important to develop molecular design methods and synthetic methods hereafter. We focus on the translation mechanism of mRNA and propose a molecular design that can promote the translation by accelerate its rate-determining step. Based on experimental data, we will explain here the strategy for the molecule design that can improve the translation efficiency and stability of mRNA.

DOI： 10.14894/medchem.31.4_194

CiNii Research

Language：English   Publisher：Angewandte Chemie International Edition  

Messenger RNAs (mRNAs) with phosphorothioate modification (PS-mRNA) to the phosphate site of A, G, C, and U with all 16 possible combinations were prepared, and the translation reaction was evaluated using an E. coli cell-free translation system. Protein synthesis from PS-mRNA increased in 12 of 15 patterns when compared with that of unmodified mRNA. The protein yield increased 22-fold when the phosphorothioate modification at A/C sites was introduced into the region from the 5′-end to the initiation codon. Single-turnover analysis of PS-mRNA translation showed that phosphorothioate modification increases the number of translating ribosomes, thus suggesting that the rate of translation initiation (rate of ribosome complex formation) is positively affected by the modification. The method provides a new strategy for improving translation by using non-natural mRNA.

DOI： 10.1002/anie.202007111

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Language：English   Publisher：Chemical Communications  

Circular RNA without a stop codon enables rolling circle translation. To produce circular RNAs, we carried out one-pot chemical synthesis of circular RNA from RNA fragments with the use of an EDC/HOBt-based chemical ligation reaction. The synthesized circular RNAs acted as translation templates, despite the presence of unnatural phosphoramidate linkages.

DOI： 10.1039/d0cc02140g

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Language：English   Publisher：Nucleosides Nucleotides and Nucleic Acids  

Eukaryotic mRNA has a cap structure at the 5′ end and a poly(A) tail at the 3′ end. The cap and poly(A) tail form a complex with multiple translation factors, and mRNA forms a circularized structure called a closed-loop model. This circularized structure reportedly not only stabilizes mRNA but also promotes ribosome recycling during translation, which improves translation efficiency. We designed an artificial mRNA that forms a circularized structure without a cap structure and poly(A) tail and found that its translational efficiency was improved compared with that of a sequence without the circularized structure in a eukaryotic translation system.

DOI： 10.1080/15257770.2019.1671593

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Language：English   Publisher：Chemical Communications  

We herein report a new approach for RNA interference, so-called "build-up RNAi" approach, where single-strand circular RNAs with a photocleavable unit or disulfide moiety were used as siRNA precursors. The advantages of using these circular RNA formats for RNAi were presented in aspects of immunogenicity and cellular uptake.

DOI： 10.1039/c9cc04872c

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Language：English   Publisher：The Pharmaceutical Society of Japan  

<p>Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (<i>i.e.</i>, disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.</p>

DOI： 10.1248/cpb.c19-00811

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Language：English   Publisher：Nucleosides Nucleotides and Nucleic Acids  

siRNA is a powerful method to suppress specific gene expression and has recently been utilized for molecular biology as well as medicine. However, introduction of dsRNA stimulates immune-responses as side-effects. In the present study, we utilized N⁶-methyl adenosine, one of the natural modified nucleosides, instead of adenosine in siRNA. When adenosine in the passenger or guide strand of siRNA was completely replaced with N⁶-methyl adenosine, the immune response against siRNA was evaded without any reduction in RNAi activity. This knowledge will promote the medical application of siRNA and enhance our understanding on cellular discrimination of non-self and self dsRNA.

DOI： 10.1080/15257770.2019.1641205

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Language：English   Publisher：Angewandte Chemie International Edition  

Development of intracellular delivery methods for antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor-ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10 minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide-modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of antisense DNA and siRNA.

DOI： 10.1002/anie.201900993

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Language：English   Publisher：Chembiochem  

Glutathione S-transferase π (GSTP <inf>1-1</inf> ) is overexpressed in many types of cancer and is involved in drug resistance. Therefore, GSTP <inf>1-1</inf> is an important target in cancer therapy, and many GST inhibitors have been reported. We had previously developed an irreversible inhibitor, GS-ESF, as an effective GST inhibitor; however, its cellular permeability was too low for it to be used in inhibiting intracellular GST. We have now developed new irreversible inhibitors by introducing sulfonyl fluoride (SF) into chloronitrobenzene (CNB). The mechanism of action was revealed to be that CNBSF first reacts with glutathione (GSH) through an aromatic substitution in the cell, then the sulfonyl group on the GSH conjugate with CNBSF reacts with Tyr108 of GST to form a sulfonyl ester bond. Our new inhibitor irreversible inhibited GSTP <inf>1-1</inf> both in vitro and in cellulo with a long duration of action.

DOI： 10.1002/cbic.201800671

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Language：English   Publisher：Methods in Molecular Biology  

This chapter describes a simple and straightforward way to obtain single-stranded circular RNA sequences in vitro. Linear RNA that is phosphorylated at the 5′ end is first prepared by a chemical or enzymatic method, then circularized using ligase. The function of the prepared circular RNA molecule, such as an ability to induce translation, can then be investigated.

DOI： 10.1007/978-1-4939-7562-4_15

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Language：English   Publisher：Nucleic Acids Research  

We developed a new approach for chemical ligation of oligonucleotides using the electrophilic phosphorothioester (EPT) group. A nucleophilic phosphorothioate group on oligonucleotides was converted into the EPT group by treatment with Sanger's reagent (1-fluoro-2,4-dinitrobenzene). EPT oligonucleotides can be isolated, stored frozen, and used for the ligation reaction. The reaction of the EPT oligonucleotide and an amino-modified oligonucleotide took place without any extra reagents at pH 7.0-8.0 at room temperature, and resulted in a ligation product with a phosphoramidate bond with a 39-85% yield. Thismethod has potential uses in biotechnology and chemical biology.

DOI： 10.1093/nar/gkx459

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

<p>In an <i>E. coli</i> cell-free translation system, we found that a circular RNA containing an infinite open reading frame produced more translation product than its linear counterpart by two orders of magnitude, because a ribosome can work more effectively towards the elongation on circular RNA than it can on linear RNA. We then tested circular RNAs containing an infinite open reading frame could be translated in eukaryotic systems, in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. We found that the circular RNAs also produced long peptides in eukaryotic translation systems, possibly owing to the rolling circle amplification mechanism.</p>

DOI： 10.2142/biophys.57.005

Open Access

CiNii Research