RESUMEN
Nonsense mutations in the coding region turn amino acid codons into termination codons, resulting in premature termination codons (PTCs). In the case of the in-frame PTC, if translation does not stop at the PTC but continues to the natural termination codon (NTC) with the insertion of an amino acid, known as readthrough, the full-length peptide is formed, albeit with a single amino acid mutation. We have previously developed the functionality-transfer oligonucleotide (FT-Probe), which forms a hybrid complex with RNA of a complementary sequence to transfer the functional group, resulting in modification of the 4-amino group of cytosine or the 6-amino group of adenine. In this study, the FT-Probe was used to chemically modify the adenosines of the PTC (UAA, UAG, and UGA) of mRNA, which were assayed for the readthrough in a reconstituted Escherichia coli translation system. The third adenosine-modified UAA produced three readthrough peptides incorporating tyrosine, glutamine and lysine at the UAA site. It should be noted that the additional modification with a cyclodextrin only induced glutamine incorporation. The adenosine modified UGA induced readthrough very efficiently with selective tryptophan incorporation. Readthrough of the modified UGA is caused by inhibition of the RF2 function. This study has demonstrated that the chemical modification of the adenosine 6-amino group of the PTC is a strategy for effective readthrough in a prokaryotic translation system.
Asunto(s)
Adenosina , Escherichia coli , Péptidos , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Adenosina/química , Adenosina/análogos & derivados , Péptidos/química , Péptidos/farmacología , Codón sin Sentido , Codón de Terminación/genética , Biosíntesis de Proteínas/efectos de los fármacosRESUMEN
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.
Asunto(s)
Fármacos Anti-VIH , Transcriptasa Inversa del VIH , VIH-1 , Mutación , Inhibidores de la Transcriptasa Inversa , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/síntesis química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/química , Fármacos Anti-VIH/síntesis química , Humanos , Relación Estructura-Actividad , Estructura Molecular , Nucleósidos/química , Nucleósidos/farmacología , Nucleósidos/síntesis química , Pruebas de Sensibilidad Microbiana , Relación Dosis-Respuesta a DrogaRESUMEN
MicroRNA(miR)-143 and miR-145 are mainly expressed in vascular smooth muscle cells. However, the relationship between plasma miR-143 or miR-145 levels and the left ventricular (LV) function in patients with heart diseases remains unclear. Blood samples were taken from the antecubital vein in patients with heart diseases (n = 52), such as coronary artery disease, old myocardial infarction, cardiomyopathy, and valvular heart disease, and controls without heart diseases (n = 22). We measured plasma miR-143 and -145 levels by quantitative RT-PCR using TaqMan MicroRNA Assays and THUNDERBIRD Probe qPCR Mix. Plasma BNP levels were also measured. Echocardiography was performed to measure the LV ejection fraction (LVEF) and LV dilation. Plasma miR-143 and miR-145 levels were significantly higher in patients with heart diseases than in controls, respectively. Plasma miR-143 and miR-145 levels were significantly higher in patients with LVEF < 50% than in those with LVEF ⧠50%, respectively. Plasma miR-143 and miR-145 levels were inversely correlated with LVEF, respectively. Plasma miR-143 and miR-145 levels were positively correlated with LV end-systolic dimension, respectively. Plasma miR-143 and -145 levels were positively correlated with plasma BNP levels, respectively. Plasma BNP levels were inversely correlated with LVEF. Plasma miR-143 and miR-145 levels are elevated in patients with LV dysfunction and may counteract LV dysfunction.
RESUMEN
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.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico , Fosfatos , Polimerasa Taq/genética , Polimerasa Taq/metabolismo , Reacción en Cadena de la Polimerasa , Mutación , NucleótidosRESUMEN
BACKGROUND: MicroRNA (miR)-143 and miR-145 are non-coding RNAs present in smooth muscle cells and the heart. However, their behavior and physiological role in patients with acute myocardial infarction (AMI) have not been clarified.MethodsâandâResults: Plasma miR-143 and miR-145 concentrations were measured on Day 0 (on admission) and on Day 7 in AMI patients who could be followed up for 6 months (n=25). The control group consisted of subjects without significant coronary stenosis (n=20). Blood samples were collected from the antecubital vein, and plasma miR-143 and miR-145 concentrations were measured by quantitative reverse transcription-polymerase chain reaction. In AMI patients (n=25), left ventricular ejection fraction (LVEF) was measured by echocardiography in the acute and chronic (6 months) phases. On Day 7, plasma miR-143 and miR-145 concentrations were significantly higher in AMI patients than in the control group and on Day 0 in AMI patients. Plasma miR-143 and miR-145 concentrations increased significantly from Day 0 to Day 7. The increase in plasma miR-143 concentrations (∆miR-143) in the acute phase was positively correlated with the increase in LVEF in the chronic phase. Among many factors, only ∆miR-143 was favorably correlated with left ventricle (LV) functional recovery in the chronic phase. CONCLUSIONS: An increase in plasma miR-143 concentrations in the acute phase may be a biomarker predicting recovery of LV function in the chronic phase in AMI patients.
Asunto(s)
MicroARNs , Infarto del Miocardio , Humanos , Volumen Sistólico , Función Ventricular Izquierda , Infarto del Miocardio/genética , Corazón , MicroARNs/genéticaRESUMEN
In nucleic acid drug discovery, it is extremely important to develop a technology to understand the distribution in target organs and to trace the degradation process in the body in order to optimize the structure and improve the efficiency of the clinical trial process. Since nucleic acid drugs are essentially metabolically degraded into numerous fragments, labeling at the internal position is preferable to that at the terminus. Due to the high molar specific activity of tritium, various approaches for tritium-labeling have been studied for nucleic acid drugs. Nevertheless, a generally-applicable method for tritium labeling of the internal position of a nucleic acid has not been established. In this study, we have demonstrated a new and efficient method for site-specific tritium labeling of the cytosine base at a predefined internal position in nucleic acid drugs. This method was developed by the chemical modification of the cytosine 4-amino group with the pyridinyl vinyl keto group by the functionality-transfer reaction using the reactive oligodeoxynucleotide (ODN), followed by reduction with NaBT4. Applicability to a variety of chemical structures, such as 5-methyl cytosine, 2'-O-methyl, 2'-fluoro ribose derivatives, Locked/Bridged nucleic acid (LNA/BNA) derivatives, as well as phosphorothioate bonds, has been evidenced using nine oligoribonucleic acid (ORN) substrates. It has been clearly demonstrated that this method is an excellent method for tritium-labeling of nucleic acid with an average conversion efficiency of 74%, an average isolated labeling yield of 60%, and an average specific activity of 61 GBq/mmol. This method is expected to contribute to the preclinical absorption, distribution, metabolism, excretion (ADME) studies of nucleic acid drug candidates.
Asunto(s)
Ácidos Nucleicos , ARN , ARN/química , Tritio , CitosinaRESUMEN
The trinucleotide CGG repeat is located in the 5'-UTR of FMR1 and its abnormal expansion and formation of a noncanonical RNA structure causes fetal genetic diseases. In this study, a small molecular dimer-type ligand consisting of dual G-clamp units for the recognition of two neighboring guanines was synthesized, and the binding properties for the r(CGG) repeats were investigated. Compound 2 was confirmed to bind to the mismatch guanines in the stem region of the r(CGG) repeat hairpin. In addition, the RNase T1 assay demonstrated that 2 induced the structural conversion of the r(CGG)8 repeat from the G-quadruplex into a hairpin-like structure.
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , G-Cuádruplex , Regiones no Traducidas 5' , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Ligandos , Repeticiones de TrinucleótidosRESUMEN
Due to the importance of the RNA chemical modifications, methods for the selective chemical modification at a predetermined site of the internal position of RNA have attracted much attention. We have developed functional artificial nucleic acids that modify a specific site of RNA in a site- and base-selective manner. In addition, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been shown to introduce additional molecules on the alkynes attached to the pyridine ring. However, it was found that some azide compounds produced the cycloadduct in lower yields. Therefore, in this study, we synthesized the pyridinyl transfer group with the alkyne attached via a polyethylene glycol (PEG) linker with a different length and optimized its structure for both the transfer and CuAAC reaction. Three new transfer groups were synthesized by introducing an alkyne group at the end of the triethylene (11), tetraethylene (12) or pentaethylen glycol linker (13) at the 5-position of the pyridine ring of (E)-3-iodo-1-(pyridin-2-yl)prop-2-en-1-one. These transfer groups were introduced to the 6-thioguanine base in the oligodeoxynucleotide (ODN) in high yields. The transfer groups 11 and 12 more efficiently underwent the cytosine modification. For the CuAAC reaction, although 7 showed low adduct yields with the anionic azide compound, the new transfer groups, especially 12 and 13, significantly improved the yields. In conclusion, the transfer groups 12 and 13 were determined to be promising compounds for the modification of long RNAs.
Asunto(s)
Azidas , ARN , Alquinos/química , Azidas/química , Oligodesoxirribonucleótidos/química , Piridinas , ARN/químicaRESUMEN
New 1,3-diazaphenoxazine derivatives (nitroG-Grasp-Guanidine, NGG) have been developed to covalently capture 8-nitro-cGMP in neutral aqueous solutions, which furnish a thiol reactive group to displace the 8-nitro group and a guanidine unit for interaction with the cyclic phosphate. The thiol group was introduced to the 1,3-diazaphenoxazine skeleton through a 2-aminobenzylthiol group (NGG-H) and its 4-methyl (NGG-pMe) and 6-methyl (NGG-oMe) substituted derivatives. The covalent adducts were formed between the NGG derivatives and 8-nitro-cGMP in neutral aqueous solutions. Among the NGG derivatives, the one with the 6-methyl group (NGG-oMe) exhibited the most efficient capture reaction. Furthermore, NGG-H showed a cell permeability into HEK-293 and RAW 264.7 cells and reduced the intracellular 8-nitro-cGMP level. The NGG derivatives developed in this study would become a valuable tool to study the intracellular role of 8-nitro-cGMP.
Asunto(s)
GMP Cíclico/análogos & derivados , Animales , GMP Cíclico/química , GMP Cíclico/metabolismo , Células HEK293 , Humanos , Ratones , Células RAW 264.7 , Análisis Espectral/métodos , AguaRESUMEN
The anthracenone ligands (1-12) with a keto-phenol and a hydroxamic acid unit were synthesized and evaluated by a restriction enzyme inhibition assay. DNA substrates composed of multiple CGCG or CGG sites are fully hydrolyzed by a restriction enzyme that is selective for each sequence. Under such conditions, the full-length DNA substrate remains only when the ligand binds to all binding sites and protects it from hydrolysis by the restriction enzymes. In the assay using AccII and the 50-mer DNA substrates containing a different number of CGCG sites at different non-binding AT base pair intervals, the more the CGCG sites, the more the full-length DNA increased. Namely, simultaneous binding of the ligand (5) to the CGCG sites increased in the order of (CGCG)5>(CGCG)2>(CGCG)1. Furthermore, the length of the spacer of the hydroxamic acid to the anthracenone skeleton played an important role in the preference for the number of the d(A/T) base pairs between the CGCG sites. The long spacer-ligand (5) showed a preference to the CGCG sites with five AT pairs, and the short spacer-ligand (10) to that with two AT pairs. The ligand (12) with the shortest spacer showed a preference in simultaneous binding to the 54-mer DNA composed of 16 continuous CGG sites in the assay using the restriction enzyme Fnu4HI that hydrolyzes the d(GCGGC)/d(CGCCG) site. Application of these ligands to biological systems including the repeat DNA sequence should be of significant interest.
Asunto(s)
Antracenos/farmacología , Enzimas de Restricción del ADN/antagonistas & inhibidores , Ácidos Hidroxámicos/farmacología , Fenoles/farmacología , Antracenos/síntesis química , Antracenos/química , Sitios de Unión/efectos de los fármacos , Enzimas de Restricción del ADN/metabolismo , Relación Dosis-Respuesta a Droga , Ácidos Hidroxámicos/química , Ligandos , Estructura Molecular , Fenoles/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
RNA higher-order structures play an important role for control of the gene expression, and the small molecules binding to these structures have potential to act as interfering agents in the RNA-mediated-pathway. In this study, we synthesized new RNA binding molecules based on the G-clamp structure and evaluated their binding properties using the model RNA. The monomeric G-clamp ligand exhibited a fluorescence quenching with RNA-binding. The dimeric G-clamp ligand showed a significant fluorescence OFF/ON response to the RNA hairpin structure containing the guanines, indicating a high affinity of the G-clamp dimer to two neighboring guanines located in the RNA hairpin loop.
Asunto(s)
Guanina/química , ARN/química , Sitios de Unión , Relación Dosis-Respuesta a Droga , Fluorescencia , Guanina/síntesis química , Ligandos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Cross-linkable 7-deaza-6-vinylguanosine (ADVP) and 7-propynyl-7-deaza-6-vinylguanosine (ADpVP) derivatives were synthesized and successfully incorporated into 2'-OMe-RNA oligonucleotides by solid-phase oligonucleotide synthesis. Analysis of their cross-link properties revealed that the 7-propynyl substituent on ADpVP induces a significant enhancement of the cross-link kinetics of the proximal 6-vinyl group to the complementary uracil base in the target RNA compared to that of ADVP. In addition, the 2'-OMe-RNA oligonucleotide containing ADpVP exhibited a higher antisense effect on luciferase production in the cell lysate than that of ADVP. These results suggested that the 7-substituted 7-deaza-6-vinylguanosine derivatives can be used as potent cross-linkers to target mRNA inside of cells.
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Guanosina/análogos & derivados , ARN/química , ARN/síntesis química , Técnicas de Química Sintética , Guanosina/química , Luciferasas/genética , Modelos Moleculares , Conformación de Ácido Nucleico , ARN/genéticaRESUMEN
Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg2+, which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat.
Asunto(s)
Cromomicina A3/metabolismo , ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Secuencia de Bases , Sitios de Unión , Cromomicina A3/química , ADN/química , Dimerización , Pruebas de Enzimas , Magnesio/química , Resonancia por Plasmón de SuperficieRESUMEN
BACKGROUND/AIM: Nitric oxide (NO) has various physiological activities. In this study, diclofenac (DF) which has a high affinity for human serum albumin (HSA) was nitrosylated to a novel NO donor (NDF). The cytotoxic effects and the mechanism of NDF were investigated. MATERIALS AND METHODS: Binding experiments of NDF to HSA were performed by the ultrafiltration method. NO was measured by the Griess method. The number of dead cells were measured using annexin V. Apoptosis and endoplasmic reticulum stress were evaluated by western blotting. RESULTS: NDF competitively inhibits the binding of DF to HSA, suggesting that NDF and DF have equivalent binding characteristics. NDF rapidly released NOx after being dissolved. At 200 µM, NDF induced cell death in human pancreatic cancer cells. Western blotting showed that NDF promoted the cleavage of PARP, caspase-3, and caspase-7. Inhibitors of caspase-1 and caspase-9 significantly suppressed NDF-induced cell death, as did a non-specific caspase inhibitor (Z-VAD). In addition, NDF significantly increased the expression of the endoplasmic reticulum stress marker, CHOP. CONCLUSION: NDF induces apoptotic cell death by causing endoplasmic reticulum stress. The findings of this study suggest that NDF may become a promising compound for the treatment of pancreatic cancer.
Asunto(s)
Apoptosis , Diclofenaco , Estrés del Retículo Endoplásmico , Donantes de Óxido Nítrico , Neoplasias Pancreáticas , Humanos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Diclofenaco/farmacología , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/metabolismo , Albúmina Sérica Humana/metabolismo , Muerte Celular/efectos de los fármacosRESUMEN
The large-scale analysis of small-molecule binding to diverse RNA structures is key to understanding the required interaction properties and selectivity for developing RNA-binding molecules toward RNA-targeted therapies. Here, we report a new system for performing the large-scale analysis of small molecule-RNA interactions using a multiplexed pull-down assay with RNA structure libraries. The system profiled the RNA-binding landscapes of G-clamp and thiazole orange derivatives, which recognizes an unpaired guanine base and are good probes for fluorescent indicator displacement (FID) assays, respectively. We discuss the binding preferences of these molecules based on their large-scale affinity profiles. In addition, we selected combinations of fluorescent indicators and different ranks of RNA based on the information and screened for RNA-binding molecules using FID. RNAs with high- and intermediate-rank RNA provided reliable results. Our system provides fundamental information about small molecule-RNA interactions and facilitates the discovery of novel RNA-binding molecules.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Biosíntesis de Proteínas , Caperuzas de ARN , Animales , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Cultivadas , Caperuzas de ARN/metabolismoRESUMEN
D-Serine is known to act as an endogenous co-agonist of the N-methyl-D-aspartate receptor in the mammalian brain and is endogenously synthesized from L-serine by a pyridoxal 5'-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the D-serine metabolism, such as serine racemase, D-amino acid oxidase, and D-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of D-serine such as a role in cell development. As part of the elucidation of the role of D-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na(+) in addition to Mg(2+) and Ca(2+), which are well-known activators for the mammalian serine racemase. Mg(2+) or Na(+) binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg(2+) and Na(+) were determined to be 1.2 µM and 2.2 mM, respectively. In the L-serine dehydrase reaction, Mg(2+) and Na(+) enhanced the k (cat) value without changing the K (m) value. Alanine mutation of the residues E207 and D213, which correspond to the Mg(2+)-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg(2+)- and Na(+)-dependent stimulation. These results suggest that Mg(2+) and Na(+) share the common metal ion-binding site.