RESUMO
Boron cluster-conjugated antisense oligonucleotides (B-ASOs) have already been developed as therapeutic agents with "two faces", namely as potential antisense inhibitors of gene expression and as boron carriers for boron neutron capture therapy (BNCT). The previously observed high antisense activity of some B-ASOs targeting the epidermal growth factor receptor (EGFR) could not be rationally assigned to the positioning of the boron cluster unit: 1,2-dicarba-closo-dodecaborane (0), [(3,3'-Iron-1,2,1',2'-dicarbollide) (1-), FESAN], and dodecaborate (2-) in the ASO chain and its structure or charge. For further understanding of this observation, we performed systematic studies on the efficiency of RNase H against a series of B-ASOs models. The results of kinetic analysis showed that pyrimidine-enriched B-ASO oligomers activated RNase H more efficiently than non-modified ASO. The presence of a single FESAN unit at a specific position of the B-ASO increased the kinetics of enzymatic hydrolysis of complementary RNA more than 30-fold compared with unmodified duplex ASO/RNA. Moreover, the rate of RNA hydrolysis enhanced with the increase in the negative charge of the boron cluster in the B-ASO chain. In conclusion, a "smart" strategy using ASOs conjugated with boron clusters is a milestone for the development of more efficient antisense therapeutic nucleic acids as inhibitors of gene expression.
Assuntos
Boro , Oligonucleotídeos Antissenso , Oligonucleotídeos Antissenso/farmacologia , Boro/metabolismo , Cinética , RNA Complementar , Ribonuclease H/genética , Ribonuclease H/metabolismo , Inativação Gênica , Oligonucleotídeos , Receptores ErbB/metabolismo , Pirimidinas , Ferro/metabolismoRESUMO
Bioassay-guided fractionation of the ethyl acetate extract from Teucrium flavum subsp. glaucum, endowed with inhibitory activity towards the HIV-1 reverse transcriptase-associated RNase H function, led to the isolation of salvigenin (1), cirsimaritin (2) and cirsiliol (3) along with the neo-clerodanes teuflavin (4) and teuflavoside (5). Acid hydrolysis of the inactive teuflavoside provided three undescribed neo-clerodanes, flavuglaucins A-C (7-9) and one known neo-clerodane (10). Among all neo-clerodanes, flavuglaucin B showed the highest inhibitory activity towards RNase H function with a IC50 value of 9.1 µM. Molecular modelling and site-directed mutagenesis analysis suggested that flavuglaucin B binds into an allosteric pocket close to RNase H catalytic site. This is the first report of clerodane diterpenoids endowed with anti-reverse transcriptase activity. Neo-clerodanes represent a valid scaffold for the development of a new class of HIV-1 RNase H inhibitors.
Assuntos
Diterpenos Clerodânicos/farmacologia , Flavonoides/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Extratos Vegetais/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H/antagonistas & inibidores , Teucrium/química , Diterpenos Clerodânicos/química , Diterpenos Clerodânicos/isolamento & purificação , Relação Dose-Resposta a Droga , Flavonoides/química , Flavonoides/isolamento & purificação , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/isolamento & purificação , Ribonuclease H/genética , Ribonuclease H/metabolismo , Relação Estrutura-AtividadeRESUMO
To assay enzyme activities and screen its inhibitors, we demonstrated a novel label-free chemiluminescent (CL) aptasensor for the sensitive detection of RNase H activity based on hairpin technology. The specific hairpin structure was a DNA-RNA chimeric strand, which contained a streptavidin aptamer sequence and a blocked RNA sequence. RNase H could specifically recognize and cleave the RNA sequence of the DNA-RNA hybrid stem, liberating the streptavidin aptamer which could be accumulated by streptavidin-coated magnetic microspheres (SA-MP). Then the CL signal was generated due to an instantaneous derivatization reaction between the specific CL reagent 3,4,5-trimethoxyphenyl-glyoxal (TMPG) and the guanine (G) nucleotides in the SA aptamer. This novel assay method exhibited a good linear relationship in the range of 0.1-10 U mL-1 under the optimized conditions. Our results suggested that the developed system was a promising platform for monitoring the RNase H activity and showed great potential in biomedical studies and drug screening.
Assuntos
Técnicas Biossensoriais/métodos , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Sequências Repetidas Invertidas , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/metabolismo , Células A549 , Regulação Alostérica , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Avaliação Pré-Clínica de Medicamentos , Estudos de Viabilidade , Humanos , Medições Luminescentes , Estreptavidina/metabolismoRESUMO
Despite the availability of several anti-retrovirals, there is still an urgent need for developing novel therapeutic strategies and finding new drugs against underexplored HIV-1 targets. Among them, there are the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) function and the cellular α-glucosidase, involved in the control mechanisms of N-linked glycoproteins formation in the endoplasmic reticulum. It is known that many natural compounds, such as pentacyclic triterpenes, are a promising class of HIV-1 inhibitors. Hence, here we tested the pentacyclic triterpene Lupeol, showing that it inhibits the HIV-1 RT-associated RNase H function. We then performed combination studies of Lupeol and the active site RNase H inhibitor RDS1759, and blind docking calculations, demonstrating that Lupeol binds to an HIV-1 RT allosteric pocket. On the bases of these results and searching for potential multitarget active drug supplement, we also investigated the anti-HIV-1 activity of Hemidesmus indicus, an Ayurveda medicinal plant containing Lupeol. Results supported the potential of this plant as a valuable multitarget active drug source. In fact, by virtue of its numerous active metabolites, H. indicus was able to inhibit not only the RT-associated RNase H function, but also the HIV-1 RT-associated RNA-dependent DNA polymerase activity and the cellular α-glucosidase.
Assuntos
Fármacos Anti-HIV/farmacologia , Inibidores Enzimáticos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Hemidesmus/química , Triterpenos Pentacíclicos/farmacologia , Ribonuclease H/antagonistas & inibidores , Sítio Alostérico , Fármacos Anti-HIV/química , Fármacos Anti-HIV/isolamento & purificação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Regulação da Expressão Gênica , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Humanos , Células Jurkat , Simulação de Acoplamento Molecular , Triterpenos Pentacíclicos/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Ribonuclease H/química , Ribonuclease H/genética , Ribonuclease H/metabolismo , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismoRESUMO
All drugs perturb the expression of many genes in the cells that are exposed to them. These gene expression changes can be divided into effects resulting from engaging the intended target and effects resulting from engaging unintended targets. For antisense oligonucleotides, developments in bioinformatics algorithms, and the quality of sequence databases, allow oligonucleotide sequences to be analyzed computationally, in terms of the predictability of their interactions with intended and unintended RNA targets. Applying these tools enables selection of sequence-specific oligonucleotides where no- or only few unintended RNA targets are expected. To evaluate oligonucleotide sequence-specificity experimentally, we recommend a transcriptomics protocol where two or more oligonucleotides targeting the same RNA molecule, but with entirely different sequences, are evaluated together. This helps to clarify which changes in cellular RNA levels result from downstream processes of engaging the intended target, and which are likely to be related to engaging unintended targets. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell- and animal models before clinical testing. Since potential adverse effects related to unintended targeting are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are especially relevant in oligonucleotide drug discovery.
Assuntos
Descoberta de Drogas/métodos , Oligonucleotídeos Antissenso/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Análise de Sequência de RNA/estatística & dados numéricos , Animais , Pareamento de Bases , Avaliação Pré-Clínica de Medicamentos , Humanos , Terapia de Alvo Molecular , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Sensibilidade e Especificidade , TermodinâmicaRESUMO
Hepatitis B virus (HBV) causes hepatitis, cirrhosis, liver failure, and liver cancer, but the current therapies that employ either nucelos(t)ide analogs or (pegylated)interferon α do not clear the infection in the large majority of patients. Inhibitors of the HBV ribonuclease H (RNaseH) that are being developed with the goal of producing anti-HBV drugs are promising candidates for use in combination with the nucleos(t)ide analogs to improve therapeutic efficacy. HBV is genetically very diverse, with at least 8 genotypes that differ by ≥8% at the sequence level. This diversity is reflected in the viral RNaseH enzyme, raising the possibility that divergent HBV genotypes or isolates may have varying sensitivity to RNaseH inhibitors. To evaluate this possibility, we expressed and purified 18 patient-derived RNaseHs from genotypes B, C, and D. Basal RNaseH activity and sensitivity to three novel RNaseH inhibitors from three different chemotypes were assessed. We also evaluated four consensus HBV RNaseHs to determine if such sequences would be suitable for use in antiviral drug screening. The patient-derived enzymes varied by over 10-fold in their basal RNaseH activities, but they were equivalently sensitive to each of the three inhibitors. Similarly, all four consensus HBV RNaseH enzymes were active and were equally sensitive to an RNaseH inhibitor. These data indicate that a wide range of RNaseH sequences would be suitable for use in antiviral drug screening, and that genotype- or isolate-specific genetic variations are unlikely to present a barrier during antiviral drug development against the HBV RNaseH.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Variação Genética , Vírus da Hepatite B/genética , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/metabolismo , Avaliação Pré-Clínica de Medicamentos , Genótipo , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/enzimologia , Hepatite B Crônica/tratamento farmacológico , Humanos , Ribonuclease H/genética , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Despite the availability of effective antiretroviral therapies, drugs for HIV-1 treatment with new mode of action are still needed. An innovative approach is aimed to identify dual HIV-1 inhibitors, small molecules that can inhibit two viral functions at the same time. Rhubarb, originated from Rheum palmatum L. and Rheum officinale Baill., is one of the earliest and most commonly used medicinal plants in Traditional Chinese Medicine (TCM) practice. We wanted to explore TCM for the identification of new chemical scaffolds with dual action abilities against HIV-1. METHODS: R. palmatum L. and R. officinale Baill. extracts along with their main single isolated constituents anthraquinone derivatives were tested on both HIV-1 Reverse Transcriptase (RT)-associated DNA Polymerase (RDDP) and Ribonuclease H (RNase H) activities in biochemical assays. Active compounds were then assayed for their effects on HIV-1 mutated RTs, integrase (IN) and viral replication. RESULTS: Both R. palmatum L. and R. officinale Baill. extracts inhibited the HIV-1 RT-associated RNase H activity. Among the isolated constituents, Sennoside A and B were effective on both RDDP and RNase H RT-associated functions in biochemical assays. Sennoside A was less potent when tested on K103N, Y181C, Y188L, N474A and Q475A mutated RTs, suggesting the involvement of two RT binding sites for its antiviral activity. Sennoside A affected also HIV-1 IN activity in vitro and HIV-1 replication in cell-based assays. Viral DNA production and time of addition studies showed that Sennoside A targets the HIV-1 reverse transcription process. CONCLUSION: Sennoside A is a new scaffold for the development of HIV-1 dual RT inhibitors.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Extratos Vegetais/farmacologia , Rheum/química , Extrato de Senna/farmacologia , Replicação Viral/efeitos dos fármacos , Antraquinonas/farmacologia , Infecções por HIV/tratamento farmacológico , Integrase de HIV/metabolismo , Transcriptase Reversa do HIV/metabolismo , Medicina Tradicional Chinesa , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H/metabolismo , Senosídeos , Especificidade da EspécieRESUMO
We have developed a novel type of biofunction-assisted, signal-turn-on sensor for simply and homogenously detecting DNA. This sensor system is composed of two types of in vitro-transcribed label-free RNAs (a 3' premature amber suppressor tRNA probe and an amber-mutated mRNA encoding a reporter protein), RNase H, and a wheat germ extract (WGE). A target DNA induces the 3' end maturation of the tRNA probe, which is enhanced by RNase H and leads to the expression of a full-length reporter protein through amber suppression in WGE, while there is almost no expression without the target due to the inactivity of the premature probe. Therefore, the target can be readily detected with the activity of the translated reporter. The catalytic reuse of the target with the help of RNase H in addition to various bioprocesses in WGE enables this sensor system to exhibit relatively high selectivity and sensitivity.
Assuntos
DNA/análise , Sondas Moleculares/metabolismo , Extratos Vegetais/química , RNA de Transferência/metabolismo , Ribonuclease H/metabolismo , Triticum/química , Biocatálise , DNA/metabolismo , Humanos , Extratos Vegetais/metabolismo , Triticum/metabolismoRESUMO
This protocol describes an extremely sensitive procedure for detecting the presence of known or unknown RNAs in a complex mixture. A selectively enriched population of RNAs is subjected to 3'-end labeling with [(32)P]pCp, and labeled products are separated from unincorporated label. The labeled RNAs are hybridized to sequence-specific complementary oligodeoxynucleotides, treated with RNase H (which cleaves RNA in an RNA-DNA hybrid) and the products analyzed by electrophoresis through denaturing polyacrylamide gels with the appropriate controls. If the RNA of interest was present and hybridized to its complementary oligonucleotide, its digestion with RNase H will result in a shift in its mobility through the gel or, if the RNA was fully degraded, its band will not appear. If the RNA of interest is not cleaved in the presence of any known complementary oligodeoxynucleotides, then its position in the gel will remain unaltered. This result may suggest the presence of a new or unknown RNA that may be identified using a variety of cloning techniques or by direct chemical sequencing methods.
Assuntos
Técnicas de Química Analítica/métodos , Marcação In Situ das Extremidades Cortadas , RNA/análise , Ribonuclease H/metabolismo , Limite de DetecçãoRESUMO
A total of 140,000 compounds were screened in a targetfree cell-based high throughput assay against HIV-1 infection, and a subset of 81 promising compounds was identified. Secondary screening of these 81 compounds revealed two putative human RNaseH2 inhibitors, RHI001 and RHI002, with IC50 value of 6.8 µM and 16 µM, respectively. RHI002 showed selective activity against human RNaseH2 while RHI001 inhibited HIV-RNaseH, E. coli RNaseH, and human RNaseH1 with IC50 value of 28.5 µM, 7.9 µM, and 31.7 µM, respectively. Kinetic analysis revealed that both inhibitors had non-competitive inhibitor-like properties. Because RNaseH2 is involved in the etiology of Aicardi-Goutier syndrome and has been suggested as an anticancer drug target, small molecule inhibitors modulating its activity would be useful for investigating the cellular function of this molecule.
Assuntos
Fármacos Anti-HIV/farmacologia , Inibidores Enzimáticos/farmacologia , HIV-1/efeitos dos fármacos , Pirimidinas/farmacologia , Ribonuclease H/antagonistas & inibidores , Tiofenos/farmacologia , Fármacos Anti-HIV/química , Doenças Autoimunes do Sistema Nervoso/tratamento farmacológico , Doenças Autoimunes do Sistema Nervoso/etiologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inibidores , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Estrutura Molecular , Malformações do Sistema Nervoso/tratamento farmacológico , Malformações do Sistema Nervoso/etiologia , Pirimidinas/química , Ribonuclease H/genética , Ribonuclease H/metabolismo , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Ribonucleases , Tiofenos/químicaRESUMO
This study is to investigate the mechanism of action of lindenane disesquiterpenoid shizukaol F on HIV-1 replication. Real time quantity PCR, ELISA assay and fluorescence methods were used to test HIV-1 reverse transcription process, RNA-dependent DNA polymerase activity, and RNase H activity, respectively. It showed that shizukaol F inhibited LTR/Gag production of HIV-1 reverse transcription with an IC50 of 9.11 micromol x L(-1). This result is consistent with its inhibitory effect on HIV-1 replication (IC50 of 6.12 micromol x L(-1)). Mechanism studies showed that compound shizukaol F inhibited HIV-1 RT-RNase H with IC50 of 26.4 micromol x L(-1), but had no effect on HIV-1 RT RNA-dependent DNA polymerase activity. In conclusion, shizukaol F is a new structural type HIV-1 RNase H inhibitor. This discovery will provide a clue for new type of reverse transcriptase inhibitors development.
Assuntos
Magnoliopsida/química , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H/antagonistas & inibidores , Sesquiterpenos/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular Tumoral , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Células HEK293 , Transcriptase Reversa do HIV/metabolismo , HIV-1/fisiologia , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Plantas Medicinais/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/isolamento & purificação , Ribonuclease H/metabolismo , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificaçãoRESUMO
A single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg(2+)-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of an Escherichia coli strain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg(2+)-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similar K(m)s for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H.
Assuntos
Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Avaliação Pré-Clínica de Medicamentos , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/genética , HIV-1/metabolismo , Magnésio/metabolismo , Modelos Moleculares , RNA de Transferência/genética , Ribonuclease H/química , Ribonuclease H/genética , Análise de Sequência de RNARESUMO
The hydroalcoholic extract obtained from the seeds of Casimiroa edulis cultivated in Sardinia (Italy) have been assayed on the two enzymatic-associated activities of the HIV-1 reverse transcriptase (RT), the RNA-dependent DNA polymerase (RDDP) and the ribonuclease H. In biochemical assays, the extract inhibited both activities in a dose-dependent manner, showing a 10-fold more potent inhibition of the HIV-1 RT RDDP activity. Furthermore, the extract was cytotoxic on K562 cell replication.
Assuntos
Casimiroa/química , Transcriptase Reversa do HIV/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Sementes/química , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/metabolismoAssuntos
DNA Antissenso/química , Sondas RNA/química , Ribonuclease H/metabolismo , Sequência de Bases , Catálise , Sistema Livre de Células , DNA Antissenso/metabolismo , Corantes Fluorescentes/química , Técnicas de Amplificação de Ácido Nucleico , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Biossíntese de Proteínas , Sondas RNA/metabolismo , RNA Mensageiro/metabolismo , Ribonuclease H/química , Triticum/metabolismoRESUMO
The essential oils of Ridolfia segetum (L.) Moris and Oenanthe crocata L. (Apiaceae), collected in Sardinia (Italy), have been assayed for two enzyme-associated activities of the HIV-1 reverse transcriptase (RT): RNA-dependent DNA polymerase (RDDP) activity and ribonuclease H (RNase H) activity. In biochemical assays, the essential oils inhibited HIV-1 RT RDDP activity in a dose-dependent manner, while they were inactive towards RNase H activity. Furthermore, the oils were cytotoxic towards K (562) cell replication. GC-MS analysis of the essential oils obtained by steam distillation of the aerial parts showed that the main components of R. segetum were alpha-phellandrene, alpha-terpinolene, beta-phellandrene, and dillapiol and those of O. crocata were sabinene, TRANS-beta-ocimene, CIS-beta-ocimene, and beta-pinene.
Assuntos
Fármacos Anti-HIV/farmacologia , Apiaceae/química , HIV-1/efeitos dos fármacos , Oenanthe/química , Óleos Voláteis/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/química , Fármacos Anti-HIV/isolamento & purificação , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fracionamento Químico , Cromatografia Gasosa-Espectrometria de Massas , HIV-1/enzimologia , Humanos , Óleos Voláteis/química , Ribonuclease H/metabolismoRESUMO
The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.
Assuntos
Hepacivirus/química , RNA Viral/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Arginina/química , Sequência de Bases , Escherichia coli/enzimologia , Hepacivirus/genética , Hidrólise , Lisina/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Ribonuclease H/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We have identified the S(MK) box as a conserved RNA motif in the 5' untranslated leader region of metK (SAM synthetase) genes in lactic acid bacteria, including Enterococcus, Streptococcus and Lactococcus species. This RNA element bound SAM in vitro, and binding of SAM caused an RNA structural rearrangement that resulted in sequestration of the Shine-Dalgarno (SD) sequence. Mutations that disrupted pairing between the SD region and a sequence complementary to the SD blocked SAM binding, whereas compensatory mutations that restored pairing restored SAM binding. The Enterococcus faecalis S(MK) box conferred translational repression of a lacZ reporter when cells were grown under conditions where SAM pools are elevated, and mutations that blocked SAM binding resulted in loss of repression, demonstrating that the S(MK) box is functional in vivo. The S(MK) box therefore represents a new SAM-binding riboswitch distinct from the previously identified S box RNAs.
Assuntos
Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/metabolismo , Regulação Bacteriana da Expressão Gênica , Metionina Adenosiltransferase/biossíntese , Metionina Adenosiltransferase/genética , Biossíntese de Proteínas/genética , S-Adenosilmetionina/metabolismo , Bacillus subtilis/genética , Sequência de Bases , Análise Mutacional de DNA , Enterococcus faecalis/genética , Genes Reporter/genética , Metionina Adenosiltransferase/química , Modelos Genéticos , Dados de Sequência Molecular , Mutação/genética , Ribonuclease H/metabolismoRESUMO
A stereoregular all-(Sp)-boranophosphate oligodeoxyribonucleotide (BH3(-)-ODN) 15-mer was synthesized using an enzymatic approach. The BH3(-)-ODN formed a hybrid with the complementary RNA 15-mer and induced RNase H hydrolysis of the RNA strand at ODN concentrations as low as 10 nM at 37 degrees C, but with a lower efficiency than that of its natural phosphodiester analogue.
Assuntos
Compostos de Boro , Oligonucleotídeos/farmacologia , Ribonuclease H/metabolismo , Sequência de Bases , Ativação Enzimática , Genes erbB-2 , Cinética , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligonucleotídeos/química , Especificidade por SubstratoRESUMO
Intracerebroventricular injections of oligonucleotide probes complementary to oxytocin mRNA are known to decrease systemic oxytocin levels. In this study we show that immunoreactive oxytocin in the magnocellular hypothalamic perikarya and in their neurohypophysial projections remains unaffected by intracerebroventricular injections with an oxytocin antisense probe in rats. Hybridization signal for oxytocin mRNA was increased in the supraoptic and paraventricular nuclei in these animals. Immunocytochemistry with a monoclonal antibody, raised against triple helical DNA resulted in an accumulation of cytoplasmic reaction product in many of the magnocellular oxytocin immunoreactive neurons and in a fraction of the Herring bodies inthe posterior pituitary lobe in the antisense treated rats. Such immunostaining could be abolished by pretreating sections with RNase H. Animals injected with a mismatch probe instead of the antisense probe were devoid of cytoplasmic or axonal triple helix immunostaining. Our findings indicate that oxytocinergic transcripts in magnocellular hypothalamic neurons form triple helix-like aggregates upon specific antisense targeting rather than being degraded by endogenous RNases. While de novo transcription of oxytocin is probably stimulated, systemic release of the nonapeptide may be impaired.
Assuntos
DNA Antissenso/imunologia , DNA Antissenso/metabolismo , DNA/imunologia , Hipotálamo/metabolismo , Neurônios/metabolismo , Ocitocina/genética , Ocitocina/metabolismo , Animais , Anticorpos Monoclonais/imunologia , DNA/genética , DNA Antissenso/genética , Imunofluorescência , Hipotálamo/citologia , Ocitocina/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ribonuclease H/metabolismo , Transcrição GênicaRESUMO
A fluorescence resonance energy transfer assay readily applicable to 96-well and 384-well microplate formats with robotic operation was developed to enable high-throughput screening for inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT)-associated RNase H activity, an underexplored target for antiretroviral development. The assay substrate is an 18-nucleotide 3'-fluorescein-labeled RNA annealed to a complementary 18-nucleotide 5'-Dabcyl-modified DNA. The intact duplex has an extremely low background fluorescent signal and provides up to 50-fold fluorescent signal enhancement following hydrolysis. The size and sequence of the duplex are such that HIV-1 RT-RNase H cuts the RNA strand close to the 3' end. The fluorescein-labeled ribonucleotide fragment readily dissociates from the complementary DNA at room temperature with immediate generation of a fluorescent signal. This assay is rapid, inexpensive, and robust, providing Z' factors of 0.8 and coefficients of variation of about 5%. The assay can be carried out both in real-time (continuous) and in "quench" modes; the latter requires only two addition steps with no washing and is thus suitable for robotic operation. Several chemical libraries totaling more than 106,000 compounds were screened with this assay in approximately 1 month.