RESUMO
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.
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Nucleoside reverse transcriptase inhibitors (NRTIs) have been extensively studied as drugs targeting HIV RT. However, the practice or use of approved NRTIs lacking the 3'-hydroxy group often promotes frequent HIV mutations and generates drug-resistance. Here, we describe a novel NRTI with 2'-ß-methylselenyl modification. We found that this modification inhibited the DNA elongation reaction by HIV-1 RT despite having a 3'-hydroxy group. Moreover, the conformation of this nucleoside analog is controlled at C3'-endo, a conformation that resists excision from the elongating DNA by HIV RT. Accordingly, the designed analogs exhibited activity against both wild-type HIV and multidrug-resistant HIV mutants.
Assuntos
Fármacos Anti-HIV , Transcriptase Reversa do HIV , HIV-1 , Mutação , Inibidores da Transcriptase Reversa , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/síntese química , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/química , Fármacos Anti-HIV/síntese química , Humanos , Relação Estrutura-Atividade , Estrutura Molecular , Nucleosídeos/química , Nucleosídeos/farmacologia , Nucleosídeos/síntese química , Testes de Sensibilidade Microbiana , Relação Dose-Resposta a DrogaRESUMO
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.
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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. OsO4 treatment followed by NH4Cl 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.
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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.
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Inativação Gênica , Oligodesoxirribonucleotídeos , RNA Mensageiro/genética , Expressão GênicaRESUMO
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 : TâG : C mutation dramatically, which is common when using Taq polymerase. Conversely, deoxyguanosine tetraphosphate (dG4P) suppressed this mutation but increased the G : CâA : T mutation during PCR. Using an excess amount of dG4P suppressed both mutations successfully and total fidelity was improved.
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Técnicas de Amplificação de Ácido Nucleico , Fosfatos , Taq Polimerase/genética , Taq Polimerase/metabolismo , Reação em Cadeia da Polimerase , Mutação , NucleotídeosRESUMO
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.
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Biossíntese de Proteínas , Capuzes de RNA , Animais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Cultivadas , Capuzes de RNA/metabolismoRESUMO
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.
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Inativação Gênica , Oligonucleotídeos , Nucleosídeos , Oligonucleotídeos/química , RNA Interferente Pequeno/química , Termodinâmica , Uridina/químicaRESUMO
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.
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Nucleotídeos , Capuzes de RNA , Células HeLa , Humanos , Biossíntese de Proteínas , Capuzes de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Synthetic phosphate-derived functional groups are important for controlling the function of bioactive molecules inâ vivo. Herein we describe the development of a new type of biocompatible phosphate analog, a fluorophosphoramidate (FPA) functional group that has characteristic P-F and P-N bonds. We found that FPA with a primary amino group was relatively unstable in aqueous solution and was converted to a monophosphate, while FPA with a secondary amino group was stable. Furthermore, by improving the molecular design of FPA, we developed a reaction in which a secondary amino group is converted to a primary amino group in the intracellular environment and clarified that the FPA group functions as a phosphate prodrug of nucleoside. Various FPA-gemcitabine derivatives were synthesized and their toxicity to cancer cells were evaluated. One of the FPA-gemcitabine derivatives showed superior toxicity compared with gemcitabine and its ProTide prodrug, which methodology is widely used in various nucleoside analogs, including anti-cancer and anti-virus drugs.
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Neoplasias , Pró-Fármacos , Antivirais/farmacologia , Humanos , Fosfatos , Pró-Fármacos/química , Pró-Fármacos/farmacologiaRESUMO
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 5â 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 24â h after addition with the amount increasing further after 48 and 72â h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72â 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 72â 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.
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Permeabilidade da Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Dissulfetos/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Núcleo Celular/metabolismo , Dissulfetos/química , Éxons , Células HeLa , Humanos , Estrutura Molecular , Fibras Musculares Esqueléticas/metabolismo , Oligonucleotídeos Antissenso/químicaRESUMO
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 258â 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.
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DNA/síntese química , DNA/química , DNA/genética , Inativação Gênica , Células HeLa , HumanosRESUMO
Significant efforts have been made to develop therapeutic RNA aptamers that exploit synthetic RNA to capture target molecules. However, ensuring RNA aptamers are resistant against intrinsic nucleases remains an issue and restricts their use as therapeutics. Introduction of chemical modifications to the 2' sugar moiety of RNA improves their stability effectively and can be achieved by chemical synthesis using modified phosphoramidites; however, this approach is not suitable for preparing long RNA molecules. Although recombinant nucleotide polymerases can transcribe RNA, these polymerases cannot synthesize modified RNA because they do not recognize 2' modified nucleoside triphosphates. In this review, we focus on several polymerase mutants that tolerate substrates containing modifications of the 2' sugar moiety to synthesize RNA, and the problems that must be overcome to prepare chemically modified RNA with high efficacy by inâ vitro transcription.
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Técnica de Seleção de AptâmerosRESUMO
The nucleosome is one of the most fundamental units involved in gene expression and consequent cell development, differentiation, and expression of cell functions. We report here a method to place reconstituted nucleosomes into a DNA origami frame for direct observation using high-speed atomic-force microscopy (HS-AFM). By using this method, multiple nucleosomes can be incorporated into a DNA origami frame and real-time movement of nucleosomes can be visualized. The arrangement and conformation of nucleosomes and the distance between two nucleosomes can be designed and controlled. In addition, four nucleosomes can be placed in a DNA frame. Multiple nucleosomes were well accessible in each conformation. Dynamic movement of the individual nucleosomes were precisely monitored in the DNA frame, and their assembly and interaction were directly observed. Neither mica surface modification nor chemical fixation of nucleosomes is used in this method, meaning that the DNA frame not only holds nucleosomes, but also retains their natural state. This method offers a promising platform for investigating nucleosome interactions and for studying chromatin structure.
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DNA , Nucleossomos , Microscopia de Força Atômica , Conformação de Ácido NucleicoRESUMO
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.
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Biossíntese de Proteínas , RNA Mensageiro/química , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Escherichia coli/metabolismo , Iniciação Traducional da Cadeia Peptídica , Oligonucleotídeos Fosforotioatos/química , RNA Mensageiro/metabolismo , Ribossomos/metabolismoRESUMO
We developed an epigenetically active, cooperative DNA binding transcription factor platform assisted by cucurbit[7]uril (CB7) host-guest modules. This new type of molecule termed ePIP-HoGu not only mimics the operation of transcription factors as a pair but also recruits the epigenetic modifier to a particular DNA locus.
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DNA/química , Epigênese Genética/genética , Fatores de Transcrição/química , Hidrocarbonetos Aromáticos com Pontes/química , DNA/genética , Imidazóis/química , Estrutura Molecular , Fatores de Transcrição/síntese química , Fatores de Transcrição/genéticaRESUMO
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.
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Eucariotos/genética , Regulação da Expressão Gênica , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Genes Reporter , Estabilidade de RNA , Coelhos , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
Electron transfer through π-stacked arrays of double-stranded DNA contributes to the redox chemistry of bases, including guanine oxidation and thymine-thymine dimer repair by photolyase. 5-Bromouracil is an attractive photoreactive thymine analogue that can be used to investigate electron transfer in DNA, and is a useful probe for protein-DNA interaction analysis. In the present study using BrU we found that UV irradiation facilitated electron injection from mitochondrial transcription factor A into DNA. We also observed that this electron injection could lead to repair of a thymine-thymine dimer.
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Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/química , DNA/química , Elétrons , Proteínas Mitocondriais/química , Dímeros de Pirimidina/química , Fatores de Transcrição/química , Sequência de Bases , Bromouracila/química , Bromouracila/efeitos da radiação , DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/efeitos da radiação , Humanos , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/efeitos da radiação , Regiões Promotoras Genéticas/efeitos da radiação , Ligação Proteica , Dímeros de Pirimidina/efeitos da radiação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/efeitos da radiação , Raios UltravioletaRESUMO
A distance- and orientation-factor-dependent FRET system is a useful and attractive approach to the investigation of the conformational dynamics of nucleosomes. In this study, the application of the highly emissive nucleobase th dG-tC FRET pair to 601â nucleosomes is reported. It was found that the th dG-tC FRET pair was successfully incorporated to 145â bp 601â sequences, and different FRET efficiencies were obtained for the designated donor and acceptor positions in the nucleosome.
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Desoxiguanosina/química , Transferência Ressonante de Energia de Fluorescência , Nucleossomos/química , Fenotiazinas/química , Pareamento de Bases , DNA/química , DNA/metabolismo , Desoxiguanosina/metabolismo , Modelos Moleculares , Nucleossomos/metabolismo , Fenotiazinas/metabolismoRESUMO
Despite evidence that histone H3 and H4 proteins may act as the precursor for orientating the DNA sequence to form nucleosome structures, there is no direct evidence of the formed compact structure. Here, it is demonstrated that a histone H3-H4 octasome could be constructed without the involvement of histone H2A-H2B under in vitro reconstitution conditions. Atomic force microscopy was used to obtain the first direct observation of the octasome structure, which exhibited a similar core-protein size as that of a nucleosome but with a shorter core histone-binding DNA region. The octasome also displayed a one-step histone-dissociation pattern under heat treatment, distinct micrococcal nuclease and peplomycin accessibility, which suggests a different wrapping pattern to that in nucleosomes.