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1.
Molecules ; 26(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670247

RESUMO

Dengue fever is one of the most common viral infections affecting humans. It is an expanding public health problem, particularly in tropical and subtropical regions. No effective vaccine or antiviral therapies against Dengue virus (DENV) infection are available. Therefore, there is a strong need to develop safe and effective therapeutic strategies that can reduce the burden and duration of hospitalizations due to this life-threatening disease. Oligonucleotide-based strategies are considered as an attractive means of inhibiting viral replication since oligonucleotides can be designed to interact with any viral RNA, provided its sequence is known. The resultant targeted destruction of viral RNA interferes with viral replication without inducing any adverse effects on cellular processes. In this review, we elaborate the ribozymes, RNA interference, CRISPR, aptamer and morpholino strategies for the inhibition of DENV replication and discuss the challenges involved in utilizing such approaches.


Assuntos
Vírus da Dengue/efeitos dos fármacos , Dengue/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Replicação Viral/efeitos dos fármacos , Antivirais/química , Antivirais/uso terapêutico , Dengue/genética , Dengue/virologia , Vírus da Dengue/patogenicidade , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , Interferência de RNA
2.
J Agric Food Chem ; 69(5): 1705-1713, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33528262

RESUMO

Multiplex and high-throughput assays are becoming the main trends in the development of new nucleic acid detection and quantification methods, such as those for genetically modified organism (GMO) analysis. Here, we report a novel universal LNA probe-mediated droplet digital polymerase chain reaction (PCR) method (ULNA-ddPCR) for multiple DNA target quantification in GMOs. In ULNA-ddPCR, only one universal LNA probe is used for multiple DNA targets instead of using one to one TaqMan probe. The specificity, sensitivity, dynamic range, and accuracy of the ULNA-ddPCR method are determined by employing GM rice analysis as an example. Simplex and triplex ULNA-ddPCR assays for three GM rice events, T2A-1, T1C-19, and G6H1, are established and evaluated. All results indicate that the developed simplex and triplex ULNA-ddPCR assays are suitable for quantitative analysis of GM rice events with high sensitivity, accuracy, and low cost. The ULNA-ddPCR method also has the potential for multiple DNA target quantification in other research fields.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Oryza/genética , Plantas Geneticamente Modificadas/genética , DNA de Plantas/genética , Oligonucleotídeos/genética , Sensibilidade e Especificidade , Zea mays
3.
Methods Mol Biol ; 2246: 69-86, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576983

RESUMO

Traditionally, RNA and DNA probes are used in fluorescence in situ hybridization (FISH) methods for microbial detection and characterization of communities' structure and diversity. However, the recent introduction of nucleic acid mimics (NAMs) has improved the robustness of the FISH methods in terms of sensitivity and specificity. Several NAMs have been used, of which the most relevant are peptide nucleic acid (PNA), locked nucleic acids (LNA), 2'-O-methyl RNA (2'OMe), and phosphorothioates (PS). In this chapter, we describe a protocol using PNA and LNA/2'OMe probes for microbial detection by FISH, pointing out the differences between them. These protocols are easily adapted to different microorganisms and different probe sequences.


Assuntos
Hibridização in Situ Fluorescente/métodos , Ácidos Nucleicos/genética , Microbiota/genética , Sondas de Ácido Nucleico/genética , Oligonucleotídeos/genética , Ácidos Nucleicos Peptídicos/genética , Sensibilidade e Especificidade
4.
Methods Mol Biol ; 2246: 157-168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576989

RESUMO

The possibility of visualizing bacteriophage-host interactions through fluorescence in situ hybridization (FISH)-derived methods is gaining relevance in the last few years. These methods allow the possibility of discriminating between phage-infected and noninfected cells and the assessment of the different infection stages at the single cell level. In opposition to bacterial cells, the detection of phages is more challenging due to the low number of nucleic acid copies. However, by using a conserved region of the phage genome that is highly expressed during transcription, a FISH signal targeting phage DNA copies and mRNA transcripts can be easily visible inside the bacterial host cells.In this book chapter, we will cover both the design of locked nucleic acid (LNA) probes for phages and a FISH method for the detection of phages inside bacterial cells.


Assuntos
Bactérias/virologia , Bacteriófagos/genética , Hibridização in Situ Fluorescente/métodos , DNA/genética , Interações entre Hospedeiro e Microrganismos/genética , Oligonucleotídeos/genética , RNA Mensageiro/genética
5.
Methods Mol Biol ; 2246: 263-277, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576995

RESUMO

Flow-Fluorescence in situ hybridization (Flow-FISH) enables multiparametric high-throughput detection of target nucleic acid sequences at the single cell-level, allowing an accurate quantification of different cell populations by using a combination of flow cytometry and fluorescent in situ hybridization (FISH). In this chapter, a flow-FISH protocol is described with labeled nucleic acid mimics (NAMs) (e.g. LNA/2'OMe and PNA) acting as the reporter molecules. This protocol allows for the specific detection of bacterial cells. Hence, this protocol can be carried out with minor adjustments, in order to simultaneously detect different species of bacteria in different types of clinical, food, or environmental samples.


Assuntos
Bactérias/genética , Hibridização in Situ Fluorescente/métodos , Sondas de Ácido Nucleico/genética , Ácidos Nucleicos/genética , Oligonucleotídeos/genética
6.
Methods Mol Biol ; 2246: 317-330, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576999

RESUMO

Currently, the interactions occurring between oligonucleotides and the cellular envelope of bacteria are not fully resolved at the molecular level. Understanding these interactions is essential to gain insights on how to improve the internalization of the tagged oligonucleotides during fluorescence in situ hybridization (FISH). Agent-based modeling (ABM) is a promising in silico tool to dynamically simulate FISH and bring forward new knowledge on this process. Notably, it is important to simulate the whole bacterial cell, including the different layers of the cell envelope, given that the oligonucleotide must cross the envelope to reach its target in the cytosol. In addition, it is also important to characterize other molecules in the cell to best emulate the cell and represent molecular crowding. Here, we review the main information that should be compiled to construct an ABM on FISH and provide a practical example of an oligonucleotide targeting the 23S rRNA of Escherichia coli .


Assuntos
Hibridização in Situ Fluorescente/métodos , Parede Celular/genética , Citosol/metabolismo , Escherichia coli/genética , Sondas de Oligonucleotídeos/genética , Oligonucleotídeos/genética , RNA Ribossômico 23S/genética
7.
Nat Commun ; 12(1): 847, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558503

RESUMO

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.


Assuntos
Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Mutação/genética , Oligonucleotídeos/química , Oligonucleotídeos/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Proteína C9orf72/química , Éxons/genética , Glutamatos/toxicidade , Íntrons/genética , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estereoisomerismo
8.
Nucleic Acids Res ; 49(2): 879-890, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33406239

RESUMO

Programmed DNA double-strand breaks (DSBs) made during meiosis are repaired by recombination with the homologous chromosome to generate, at selected sites, reciprocal crossovers that are critical for the proper separation of homologs in the first meiotic division. Backup repair processes can compensate when the normal meiotic recombination processes are non-functional. We describe a novel backup repair mechanism that occurs when the homologous chromosome is not available in Drosophila melanogaster meiosis. In the presence of a previously described mutation (Mcm5A7) that disrupts chromosome pairing, DSB repair is initiated by homologous recombination but is completed by non-homologous end joining (NHEJ). Remarkably, this process yields precise repair products. Our results provide support for a recombination intermediate recently proposed in mouse meiosis, in which an oligonucleotide bound to the Spo11 protein that catalyzes DSB formation remains bound after resection. We propose that this oligonucleotide functions as a primer for fill-in synthesis to allow scarless repair by NHEJ. We argue that this is a conserved repair mechanism that is likely to be invoked to overcome occasional challenges in normal meiosis.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Meiose/genética , Oligonucleotídeos/genética , Animais , Proteínas de Ciclo Celular/genética , Simulação por Computador , Troca Genética , DNA Ligase Dependente de ATP/fisiologia , Proteínas de Drosophila/genética , Endodesoxirribonucleases/fisiologia , Feminino , Masculino , Modelos Genéticos , Mutação de Sentido Incorreto , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Rad51 Recombinase/fisiologia , Alinhamento de Sequência , Deleção de Sequência , Sequenciamento Completo do Genoma
9.
Int J Biol Macromol ; 173: 34-43, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33476618

RESUMO

The prion protein (PrP) misfolding to its infectious form is critical to the development of prion diseases, whereby various ligands are suggested to participate, such as copper and nucleic acids (NA). The PrP globular domain was shown to undergo NA-driven liquid-liquid phase separation (LLPS); this latter may precede pathological aggregation. Since Cu(II) is a physiological ligand of PrP, we argue whether it modulates phase separation altogether with nucleic acids. Using recombinant PrP, we investigate the effects of Cu(II) (at 6 M equivalents) and a previously described PrP-binding GC-rich DNA (equimolarly to protein) on PrP conformation, oligomerization, and phase transitions using a range of biophysical techniques. Raman spectroscopy data reveals the formation of the ternary complex. Microscopy suggests that phase separation is mainly driven by DNA, whereas Cu(II) has no influence. Our results show that DNA can be an adjuvant, leading to the structural conversion of PrP, even in the presence of an endogenous ligand, copper. These results provide new insights into the role of Cu(II) and NA on the phase separation, structural conversion, and aggregation of PrP, which are critical events leading to neurodegeneration.


Assuntos
Cobre/química , Oligonucleotídeos/química , Proteínas da Gravidez/química , Agregados Proteicos , Animais , Cátions Bivalentes , Clonagem Molecular , Cobre/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Camundongos , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
PLoS One ; 16(1): e0238753, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481821

RESUMO

PFRED a software application for the design, analysis, and visualization of antisense oligonucleotides and siRNA is described. The software provides an intuitive user-interface for scientists to design a library of siRNA or antisense oligonucleotides that target a specific gene of interest. Moreover, the tool facilitates the incorporation of various design criteria that have been shown to be important for stability and potency. PFRED has been made available as an open-source project so the code can be easily modified to address the future needs of the oligonucleotide research community. A compiled version is available for downloading at https://github.com/pfred/pfred-gui/releases/tag/v1.0 as a java Jar file. The source code and the links for downloading the precompiled version can be found at https://github.com/pfred.


Assuntos
Biologia Computacional/métodos , Primers do DNA/genética , Oligonucleotídeos Antissenso/genética , Algoritmos , Biblioteca Gênica , Genômica , Oligonucleotídeos/genética , RNA Interferente Pequeno/genética , Software , Interface Usuário-Computador
11.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430158

RESUMO

(1) Background: Chiral nanoparticular systems have recently emerged as a compelling platform for investigating stereospecific behavior at the nanoscopic level. We describe chiroselective supramolecular interactions that occur between DNA oligonucleotides and chiral polyurea nanocapsules. (2) Methods: We employ interfacial polyaddition reactions between toluene 2,4-diisocyanate and lysine enantiomers that occur in volatile oil-in-water nanoemulsions to synthesize hollow, solvent-free capsules with average sizes of approximately 300 nm and neutral surface potential. (3) Results: The resultant nanocapsules exhibit chiroptical activity and interact differentially with single stranded DNA oligonucleotides despite the lack of surface charge and, thus, the absence of significant electrostatic interactions. Preferential binding of DNA on D-polyurea nanocapsules compared to their L-counterparts is demonstrated by a fourfold increase in capsule size, a 50% higher rise in the absolute value of negative zeta potential (ζ-potential), and a three times lower free DNA concentration after equilibration with the excess of DNA. (4) Conclusions: We infer that the chirality of the novel polymeric nanocapsules affects their supramolecular interactions with DNA, possibly through modification of the surface morphology. These interactions can be exploited when developing carriers for gene therapy and theranostics. The resultant constructs are expected to be highly biocompatible due to their neutral potential and biodegradability of polyurea shells.


Assuntos
DNA/química , Portadores de Fármacos/farmacologia , Nanocápsulas/química , Oligonucleotídeos/química , Aptâmeros de Nucleotídeos/química , DNA/antagonistas & inibidores , Portadores de Fármacos/química , Emulsões/química , Emulsões/farmacologia , Humanos , Oligonucleotídeos/genética , Tamanho da Partícula , Polímeros/química
12.
Methods Mol Biol ; 2234: 297-309, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33165794

RESUMO

Recombinant expression of a gene of interest is a convenient method to study the function of the encoded protein. To introduce a gene into an expression vector, it is necessary to analyze the structure of the gene and determine some of its basic features including the exact start and end of the coding region or its exon/intron boundaries. Based on this gene analysis, oligonucleotides are then designed for amplification of the coding region by PCR and insertion into the expression vector. For the design of these oligonucleotides, the coding region of the gene of interest and the choice of expression vector must be considered. Here, a number of basic techniques for gene analysis and oligonucleotide design based on specific features of expression vectors are discussed, as well as different methods for introduction of the amplified gene into the expression vector.


Assuntos
Simulação por Computador , Expressão Gênica , Técnicas Genéticas , Vetores Genéticos/metabolismo , Oligonucleotídeos/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética
13.
Methods Mol Biol ; 2189: 19-30, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180290

RESUMO

The success of any oligonucleotide-based experiment strongly depends on the accurate design of the components. Oli2go is a user-friendly web tool that provides efficient multiplex oligonucleotide design including specificity and primer dimer checks. Its fully automated workflow involves important design steps that use specific parameters to produce high-quality oligonucleotides. This chapter describes how these steps are computationally implemented by oli2go.


Assuntos
Algoritmos , Oligonucleotídeos/genética , Análise de Sequência de DNA , Software
14.
Methods Mol Biol ; 2198: 287-299, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822039

RESUMO

Ligation of a hairpin oligonucleotide to genomic DNA prior to bisulfite conversion and PCR amplification physically links the two complementary DNA strands. This additional step in the conversion procedure overcomes the limitations of conventional bisulfite sequencing where information of the cytosine methylation status is only obtained from one of the two strands of an individual DNA molecule. Sequences derived from hairpin bisulfite PCR products reveal the dynamics of this epigenetic memory system on both strands of individual DNA molecules. The chapter describes a reliable step-by-step procedure to generate hairpin-linked DNA. It also provides a guide for efficient bisulfite conversion that is suitable for both conventional and hairpin bisulfite sequencing approaches.


Assuntos
Sequências Repetidas Invertidas/genética , Reação em Cadeia da Polimerase/métodos , Sulfitos/química , Citosina , DNA/genética , Metilação de DNA , DNA Complementar/química , DNA Complementar/genética , Humanos , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos/genética , Análise de Sequência de DNA/métodos
15.
Nucleic Acids Res ; 49(1): 479-490, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330934

RESUMO

The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.


Assuntos
Processamento Alternativo/genética , Aminoaciltransferases/genética , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Dobramento de RNA , Precursores de RNA/genética , RNA Mensageiro/genética , Células A549 , Sequência de Bases , Linhagem Celular Tumoral , Sequência Conservada , Éxons/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/genética , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/genética , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Elongação da Transcrição Genética
16.
Methods Mol Biol ; 2167: 79-89, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712916

RESUMO

Deoxyribozymes capable of catalyzing sequence-specific RNA cleavage have broad applications in biotechnology. In vitro selected RNA-cleaving deoxyribozymes normally contain two substrate-binding arms and a central catalytic core region. Here, we describe the systematic characterization and optimization of an RNA-cleaving deoxyribozyme with an unusually short left binding arm, and its special sequence requirement for its optimal catalytic activity.


Assuntos
DNA Catalítico/química , DNA Catalítico/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Conformação de Ácido Nucleico , Clivagem do RNA/efeitos dos fármacos , Sequência de Bases , Catálise , Domínio Catalítico , DNA Catalítico/genética , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Íons/química , Cinética , Metais/química , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/genética , Clivagem do RNA/genética , Especificidade por Substrato
17.
ACS Appl Mater Interfaces ; 13(1): 207-218, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33348979

RESUMO

Functional core/shell particles are highly sought after in analytical chemistry, especially in methods suitable for single-particle analysis such as flow cytometry because they allow for facile multiplexed detection of several analytes in a single run. Aiming to develop a powerful bead platform of which the core particle can be doped in a straightforward manner while the shell offers the highest possible sensitivity when functionalized with (bio)chemical binders, polystyrene particles were coated with different kinds of mesoporous silica shells in a convergent growth approach. Mesoporous shells allow us to obtain distinctly higher surface areas in comparison with conventional nonporous shells. While assessing the potential of narrow- as well as wide-pore silicas such as Mobil composition of matter no. 41 (MCM-41) and Santa Barbara amorphous material no. 15 (SBA-15), especially the synthesis of the latter shells that are much more suitable for biomolecule anchoring was optimized by altering the pH and both, the amount and type of the mediator salt. Our studies showed that the best performing material resulted from a synthesis using neutral conditions and MgSO4 as an ionic mediator. The analytical potential of the particles was investigated in flow cytometric DNA assays after their respective functionalization for individual and multiplexed detection of short oligonucleotide strands. These experiments revealed that a two-step modification of the silica surface with amino silane and succinic anhydride prior to coupling of an amino-terminated capture DNA (c-DNA) strand is superior to coupling carboxylic acid-terminated c-DNA to aminated core/shell particles, yielding limits of detection (LOD) down to 5 pM for a hybridization assay, using labeled complementary single-stranded target DNA (t-DNA) 15mers. The potential of the use of the particles in multiplexed analysis was shown with the aid of dye-doped core particles carrying a respective SBA-15 shell. Characteristic genomic sequences of human papillomaviruses (HPV) were chosen as the t-DNA analytes here, since their high relevance as carcinogens and the high number of different pathogens is a relevant model case. The title particles showed a promising performance and allowed us to unequivocally detect the different high- and low-risk HPV types in a single experimental run.


Assuntos
DNA Viral/análise , Citometria de Fluxo/métodos , Microplásticos/química , Poliestirenos/química , Dióxido de Silício/química , Alphapapillomavirus/química , Compostos de Boro/química , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/genética , DNA Viral/genética , Fluoresceínas/química , Corantes Fluorescentes/química , Limite de Detecção , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos/genética , Porosidade
18.
J Clin Virol ; 131: 104581, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32889496

RESUMO

INTRODUCTION: During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing these tests for broad, long-term detection was complicated by limited information about the novel virus' genome sequence and how it might mutate during global spread and adaptation to humans. METHODS: We assessed eight widely adopted lab developed PCR tests for SARS-CoV-2 against 15,001 SARS-CoV-2 genome sequences. Using a custom bioinformatic pipeline called PCR_strainer, we identified all mismatches and sequence variants in genome locations targeted by 15 sets of primer/probe oligonucleotides from these assays. RESULTS: For 12 out of 15 primer/probe sets, over 98 % of SARS-CoV-2 genomes had no mismatches. Two primer/probe sets contained a single mismatch in the reverse primer that was present in over 99 % of genomes. One primer/probe set targeted a location with extensive polymorphisms with 23 sequence observed variants at the forward primer location. One of these variants, which contains three nucleotide mismatches, arose in February as part of the emergence of a viral clade and was present in 18.8 % of the genomes we analyzed. DISCUSSION: Most early PCR diagnostic tests for SARS-CoV-2 remain inclusive of circulating viral diversity, but three assays with extensive mismatches highlight assay design challenges for novel pathogens and provide valuable lessons for PCR assay design during future outbreaks. Our bioinformatics pipeline is also presented as a useful general-purpose tool for assessing PCR diagnostics assays against circulating strains.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Oligonucleotídeos/genética , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Biologia Computacional , Simulação por Computador , Infecções por Coronavirus/virologia , Genoma Viral , Humanos , Pandemias , Pneumonia Viral/virologia , RNA Viral , Sensibilidade e Especificidade
19.
Nucleic Acids Res ; 48(18): 10555-10566, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32890406

RESUMO

A hybrid-type G-quadruplex is modified with LNA (locked nucleic acid) and 2'-F-riboguanosine in various combinations at the two syn positions of its third antiparallel G-tract. LNA substitution in the central tetrad causes a complete rearrangement to either a V-loop or antiparallel structure, depending on further modifications at the 5'-neighboring site. In the two distinct structural contexts, LNA-induced stabilization is most effective compared to modifications with other G surrogates, highlighting a potential use of LNA residues for designing not only parallel but various more complex G4 structures. For instance, the conventional V-loop is a structural element strongly favored by an LNA modification at the V-loop 3'-end in contrast with an alternative V-loop, clearly distinguishable by altered conformational properties and base-backbone interactions as shown in a detailed analysis of V-loop structures.


Assuntos
DNA/ultraestrutura , Quadruplex G , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , DNA/química , Guanosina/genética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oligonucleotídeos/química , Termodinâmica
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