RESUMO
More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/genética , COVID-19/genética , Teste de Ácido Nucleico para COVID-19/economia , Teste para COVID-19/métodos , Proteínas do Nucleocapsídeo de Coronavírus/genética , Primers do DNA , Eletroforese em Gel de Ágar/métodos , Humanos , Laboratórios , Nasofaringe/virologia , RNA Viral/genética , Ribonuclease P/genética , Ribonuclease P/metabolismo , SARS-CoV-2/patogenicidade , Sensibilidade e EspecificidadeRESUMO
Ribonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5' tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5' leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.
Assuntos
Antraquinonas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Ribonuclease P/antagonistas & inibidores , Antraquinonas/química , Antraquinonas/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Corantes Fluorescentes , Fluorometria , Hematoxilina/análogos & derivados , Hematoxilina/química , Hematoxilina/metabolismo , Hematoxilina/farmacologia , Simulação de Dinâmica Molecular , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Ribonuclease P/química , Ribonuclease P/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
RNase P is a ribozyme originally identified for its role in maturation of tRNAs by cleavage of precursor tRNAs (pre-tRNAs) at the 5'-end termini. RNase P is a ribonucleoprotein consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. The site of cleavage of a pre-tRNA is identified by its tertiary structure; and any RNA molecule can be cleaved by RNase P as long as the RNA forms a duplex that resembles the regional structure in the pre-tRNA. When the antisense sequence that forms the duplex with the strand that is subsequently cleaved by RNase P is in a separate molecule, it is called an external guide sequence (EGS). These fundamental observations are the basis for EGS technology, which consists of inhibiting gene expression by utilizing an EGS that elicits RNase P-mediated cleavage of a target mRNA molecule. EGS technology has been used to inhibit expression of a wide variety of genes, and may help development of novel treatments of diseases, including multidrug-resistant bacterial and viral infections.
Assuntos
Oligorribonucleotídeos Antissenso/metabolismo , RNA Bacteriano/metabolismo , RNA Catalítico/metabolismo , Ribonuclease P/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Infecções Bacterianas/prevenção & controle , Humanos , Modelos Genéticos , Conformação de Ácido Nucleico , Oligorribonucleotídeos Antissenso/genética , RNA Bacteriano/química , RNA Bacteriano/genéticaRESUMO
EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Divisão Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Desenho de Fármacos , Oligorribonucleotídeos Antissenso/farmacologia , Clivagem do RNA/efeitos dos fármacos , Ribonuclease P/metabolismo , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli , Microscopia Confocal , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Regiões Terminadoras Genéticas/genéticaRESUMO
L1Tc is a non-LTR LINE element from Trypanosoma cruzi that encodes its transposition machinery and bears an internal promoter. Herewith, we report the identification of an in vitro active hepatitis delta virus-like ribozyme located in the first 77 nt at the 5'-end of the L1Tc mRNA (L1TcRz). The data presented show that L1TcRz has a co-transcriptional function. Using gel-purified uncleaved RNA transcripts, the data presented indicate that the kinetics of the self-cleaving, in a magnesium-dependent reaction, fits to a two-phase decay curve. The cleavage point identified by primer extension takes place at +1 position of the element. The hydroxyl nature of the 5'-end of the 3'-fragment generated by the cleavage activity of L1TcRz was confirmed. Since we have previously described that the 77-nt long fragment located at the 5'-end of L1Tc has promoter activity, the existence of a ribozyme in L1Tc makes this element to be the first described non-LTR retroelement that has an internal promoter-ribozyme dual function. The L1Tc nucleotides located downstream of the ribozyme catalytic motif appear to inhibit its activity. This inhibition may be influenced by the existence of a specific L1Tc RNA conformation that is recognized by RNase P.
Assuntos
Elementos Nucleotídeos Longos e Dispersos , RNA Catalítico/química , RNA Catalítico/genética , RNA Mensageiro/química , Trypanosoma cruzi/genética , Regiões 5' não Traduzidas , Sequência de Bases , Domínio Catalítico , Vírus Delta da Hepatite/enzimologia , Cinética , Dados de Sequência Molecular , Clivagem do RNA , Dobramento de RNA , RNA Catalítico/metabolismo , Ribonuclease P/metabolismo , Transcrição GênicaRESUMO
Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6')-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6')-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6')-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.