RESUMO
High-precision viral detection at point of need with clinical samples plays a pivotal role in the diagnosis of infectious diseases and the control of a global pandemic. However, the complexity of clinical samples that often contain very low viral concentrations makes it a huge challenge to develop simple diagnostic devices that do not require any sample processing and yet are capable of meeting performance metrics such as very high sensitivity and specificity. Herein we describe a new single-pot and single-step electrochemical method that uses real-time kinetic profiling of the interaction between a high-affinity aptamer and an antigen on a viral surface. This method generates many data points per sample, which when combined with machine learning, can deliver highly accurate test results in a short testing time. We demonstrate this concept using both SARS-CoV-2 and Influenza A viruses as model viruses with specifically engineered high-affinity aptamers. Utilizing this technique to diagnose COVID-19 with 37 real human saliva samples results in a sensitivity and specificity of both 100 % (27 true negatives and 10 true positives, with 0 false negative and 0 false positive), which showcases the superb diagnostic precision of this method.
Assuntos
Aptâmeros de Nucleotídeos , COVID-19 , Técnicas Eletroquímicas , Aprendizado de Máquina , SARS-CoV-2 , Aptâmeros de Nucleotídeos/química , Humanos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/imunologia , Técnicas Eletroquímicas/métodos , COVID-19/diagnóstico , COVID-19/virologia , Cinética , Vírus da Influenza A , Antígenos Virais/análise , Antígenos Virais/imunologia , Técnicas Biossensoriais/métodosRESUMO
Functional nucleic acids (FNAs), such as DNAzymes and DNA aptamers, can be engineered into circular forms for improved performance. Circular FNAs are promising candidates for bioanalytical and biomedical applications due to their intriguing properties of enhanced biological stability and compatibility with rolling circle amplification. They are typically made from linear single-stranded (ss) DNA molecules via ligase-mediated ligation. However, it remains a great challenge to synthesize circular ssDNA molecules in high yield due to inherent side reactions where two or more of the same ssDNA molecules are ligated. Herein, we present a strategy to overcome this issue by first using in vitro selection to search from a random-sequence DNA library a ligatable DNA aptamer that binds a DNA ligase and then by engineering this aptamer into a general-purpose templating DNA scaffold to guide the ligase to execute selective intramolecular circularization. We demonstrate the broad utility of this approach via the creation of several species of circular DNA molecules, including a circular DNAzyme sensor for a bacterium and a circular DNA aptamer sensor for a protein target with excellent detection sensitivity and specificity.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA Catalítico , DNA Catalítico/metabolismo , Aptâmeros de Nucleotídeos/química , Ligases/metabolismo , Técnicas de Amplificação de Ácido Nucleico , DNA/química , DNA Circular , DNA de Cadeia SimplesRESUMO
Clostridium difficile frequently causes an infectious disease known as Clostridium difficile infection (CDI), and there is an urgent need for the development of more effective rapid diagnostic tests for CDI. Previously we have developed an RNA-cleaving fluorogenic DNAzyme (RFD) probe, named RFD-CD1, that is capable of detecting a specific strain of C.â difficile but is too specific to recognize other pathogenic C.â difficile strains. To overcome this issue, herein we report RFD-CD2, another RFD that is not only highly specific to C.â difficile but also capable of recognizing diverse pathogenic C.â difficile strains. Extensive sequence and structure characterization establishes a pseudoknot structure and a significantly minimized sequence for RFD-CD2. As a fluorescent sensor, RFD-CD2 can detect C.â difficile at a concentration as low as 100â CFU/mL, thus making this DNAzyme an attractive molecular probe for rapid diagnosis of CDI caused by diverse strains of C.â difficile.
Assuntos
Clostridioides difficile , Infecções por Clostridium , DNA Catalítico , Humanos , Clostridioides difficile/genética , Infecções por Clostridium/diagnóstico , Testes de Diagnóstico RápidoRESUMO
A new method for the detection of genomic RNA combines RNA cleavage by the 10-23 DNAzyme and use of the cleavage fragments as primers to initiate rolling circle amplification (RCA). 230 different 10-23 DNAzyme variants were screened to identify those that target accessible RNA sites within the highly structured RNA transcripts of SARS-CoV-2. A total of 28 DNAzymes were identified with >20 % cleavage, 5 with >40 % cleavage and one with >60 % in 10â min. The cleavage fragments from these reactions were then screened for coupling to an RCA reaction, leading to the identification of several cleavage fragments that could efficiently initiate RCA. Using a newly developed quasi-exponential RCA method with a detection limit of 500 aM of RNA, 14 RT-PCR positive and 15 RT-PCR negative patient saliva samples were evaluated for SARS-CoV-2 genomic RNA, achieving a clinical sensitivity of 86 % and specificity of 100 % for detection of the virus in <2.5â h.
Assuntos
Técnicas Biossensoriais , COVID-19 , DNA Catalítico , Humanos , DNA Catalítico/metabolismo , RNA , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Clivagem do RNA , COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Genômica , Técnicas Biossensoriais/métodosRESUMO
We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.
Assuntos
Aptâmeros de Nucleotídeos , COVID-19/virologia , Biblioteca Gênica , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Sequência de Bases , COVID-19/diagnóstico , Colorimetria/métodos , Humanos , Conformação de Ácido Nucleico , Técnica de Seleção de AptâmerosRESUMO
Fusobacterium nucleatum has been correlated to many poor human conditions including oral infections, adverse pregnancies and cancer, and thus molecular tools capable of detecting this human pathogen can be used to develop diagnostic tests for them. Using a new selection method targeting thermally stable proteins without a counter-selection step, we derived an fluorogenic RNA-cleaving DNAzyme, named RFD-FN1, that can be activated by a thermally stable protein target that is unique to F. nucleatum subspecies. High thermal stability of protein targets is a very desirable attribute for DNAzyme-based biosensing directly with biological samples because nucleases found inherently in these samples can be heat-inactivated. We further demonstrate that RFD-FN1 can function as a fluorescent sensor in both human saliva and human stool samples. The discovery of RFD-FN1 paired with a highly thermal stable protein target presents opportunities for developing simpler diagnostic tests for this important pathogen.
Assuntos
DNA Catalítico , Fusobacterium nucleatum , Gravidez , Feminino , Humanos , DNA Catalítico/metabolismo , RNA/metabolismoRESUMO
Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd-Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 103 copies/mL. The ELABA was also used to test 50 SARS-CoV-2-positive and 60 SARS-CoV-2-negative patient saliva samples, providing sensitivity and specificity values of 84.0 and 98.3%, respectively, thus highlighting the potential of TMSA52 for the development of future rapid tests.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , Glicoproteína da Espícula de Coronavírus , Bioensaio , OligonucleotídeosRESUMO
Invited for the cover of this issue are Johnâ Brennan, Yingfuâ Li, and co-workers at McMaster University. The image depicts MSA52 as a universal DNA aptamer that recognizes spike proteins of diverse SARS-CoV-2 variants of concern. Read the full text of the article at 10.1002/chem.202200078.
RESUMO
We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed Kd values ranging from 2 to 10â nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with Kd values between 20 and 50â pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.
Assuntos
Aptâmeros de Nucleotídeos , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , COVID-19/virologia , Humanos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
We report a simple and rapid saliva-based SARS-CoV-2 antigen test that utilizes a newly developed dimeric DNA aptamer, denoted as DSA1N5, that specifically recognizes the spike proteins of the wildtype virus and its Alpha and Delta variants with dissociation constants of 120, 290 and 480â pM, respectively, and binds pseudotyped lentiviruses expressing the wildtype and alpha trimeric spike proteins with affinity constants of 2.1â pM and 2.3â pM, respectively. To develop a highly sensitive test, DSA1N5 was immobilized onto gold electrodes to produce an electrochemical impedance sensor, which was capable of detecting 1000 viral particles per mL in 1:1 diluted saliva in under 10â min without any further sample processing. Evaluation of 36 positive and 37 negative patient saliva samples produced a clinical sensitivity of 80.5 % and specificity of 100 % and the sensor could detect the wildtype virus as well as the Alpha and Delta variants in the patient samples, which is the first reported rapid test that can detect any emerging variant of SARS-CoV-2.
Assuntos
Antígenos Virais/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Teste Sorológico para COVID-19 , Técnicas Eletroquímicas , SARS-CoV-2/genética , Humanos , Saliva/químicaRESUMO
Two DNA aptamers that bind the heparin-binding domain (HBD) of the human vascular endothelial growth factor 165 (VEGF-165) have been previously reported. Although VEGF-165 is a homodimeric protein and the two aptamers have different sequences and secondary structures, the aptamers appear to occupy the same binding site and cannot form a 2 : 1 aptamer/protein complex, thus making them unsuitable for creating a higher-affinity dimeric DNA aptamer. This has motivated us to conduct a new inâ vitro selection experiment to search for new VEGF-165-binding DNA aptamers with different properties. We undertook a multistream selection strategy in which the concentration of VEGF-165 was varied significantly. We carried out 11â rounds of selection, and next-generation sequencing was conducted for every round in each stream. From comprehensive sequence analysis, we identified four classes of DNA aptamers, of which two were reported before, but two are new DNA aptamers. One of the new aptamers exhibits a unique property that has never been observed before: it is capable of forming the 2 : 1 aptamer/protein complex with VEGF-165. This work has expanded the repertoire of VEGF-165-binding DNA aptamers and creates a possibility to engineer a higher affinity homodimeric aptamer for VEGF-165.
Assuntos
Aptâmeros de Nucleotídeos/química , Fator A de Crescimento do Endotélio Vascular/química , Aptâmeros de Nucleotídeos/genética , Sítios de Ligação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Herein, we report on the design of a programmable DNA ribbon using long-chain DNA molecules with a user-defined repetitive padlock sequence. The DNA ribbon can be further combined with gold nanoparticles (AuNPs) to create a composite nanomaterial that contains an AuNP core and a high-density DNA crown carrying a cancer-cell-targeting DNA aptamer, a fluorescent tag for location tracking, and a cell-killing drug. This composite material can be efficiently internalized by cancer cells and its cellular location can be tracked by fluorescence imaging. The system offers several attractive characteristics, including simple design, tunable DNA crown, high drug-loading capacity, selective cell targeting, and pH-sensitive drug release. These features make such a material a promising therapeutic agent.
Assuntos
Antineoplásicos/administração & dosagem , DNA/química , Sistemas de Liberação de Medicamentos , Ouro/química , Nanopartículas Metálicas/química , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Humanos , Microscopia de Força AtômicaRESUMO
DNA is a highly programmable material that can be configured into unique high-order structures, such as DNA branched junctions containing multiple helical arms converging at a center. Herein we show that DNA programmability can deliver inâ situ growth of a 3-way junction-based DNA structure (denoted Y-shaped DNA) with the use of three hairpin-shaped DNA molecules as precursors, a specific microRNA target as a recyclable trigger, and a DNA polymerase as a driver. We demonstrate that the Y-shaped configuration comes with the benefit of restricted freedom of movement in confined cellular environment, which makes the approach ideally suited for inâ situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence inâ situ hybridization (FISH) method and an analogous amplified imaging method via programmed growth of a double-stranded DNA (rather than Y-shaped DNA) product.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , MicroRNAs/análise , Biocatálise , DNA Polimerase Dirigida por DNA/química , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Células MCF-7 , MicroRNAs/metabolismo , Imagem ÓpticaRESUMO
DNA catenanes are assemblies made up of two or more DNA rings linked together through mechanical bonds, and they are desirable for engineering unique nanoscale devices. However, current methods of synthesizing DNA catenanes rely on the formation of strong linking duplexes between component units to enable interlocking and thus do not permit the synthesis of complex single-stranded DNA structures with freely functioning units. We have recently reported DNA sequences that can thread through a DNA circle without the formation of a linking duplex. Here we show that these unique DNA molecules can be further used to make intricate symmetric or asymmetric DNA [3]catenanes, single-stranded DNA assemblies made up of a central mother ring interlocked to two identical or fraternal twin daughter rings, which have never been reported before. These addressable freely functioning interlocked DNA rings should facilitate the design of elaborate nanoscale machines based on DNA.
Assuntos
DNA Catenado/química , Enzimas de Restrição do DNA , DNA Catenado/síntese química , DNA Catenado/metabolismo , Eletroforese em Gel de Poliacrilamida , Nanoestruturas/química , Técnicas de Amplificação de Ácido NucleicoRESUMO
Pathogenic strains of bacteria are known to cause various infectious diseases and there is a growing demand for molecular probes that can selectively recognize them. Here we report a special DNAzyme (catalytic DNA), RFD-CD1, that shows exquisite specificity for a pathogenic strain of Clostridium difficile (C. difficile). RFD-CD1 was derived by an inâ vitro selection approach where a random-sequence DNA library was allowed to react with an unpurified molecular mixture derived from this strain of C. difficle, coupled with a subtractive selection strategy to eliminate cross-reactivities to unintended C. difficile strains and other bacteria species. RFD-CD1 is activated by a truncated version of TcdC, a transcription factor, that is unique to the targeted strain of C. difficle. Our study demonstrates for the first time that inâ vitro selection offers an effective approach for deriving functional nucleic acid probes that are capable of achieving strain-specific recognition of bacterial pathogens.
Assuntos
Clostridioides difficile/patogenicidade , DNA Catalítico/metabolismoRESUMO
Rolling circle amplification (RCA) has been widely used as an isothermal DNA amplification technique for diagnostic and bioanalytical applications. Because RCA involves repeated copying of the same circular DNA template by a DNA polymerase thousands of times, we hypothesized there exist DNA sequences that can function as optimal templates and produce more DNA amplicons within an allocated time. Herein we describe an in vitro selection effort conducted to search from a random sequence DNA pool for such templates for phi29 DNA polymerase, a frequently used polymerase for RCA. Diverse DNA molecules were isolated and they were characterized by richness in adenosine (A) and cytidine (C) nucleotides. The top ranked sequences exhibit superior RCA efficiency and the use of these templates for RCA results in significantly improved detection sensitivity. AC-rich sequences are expected to find useful applications for setting up effective RCA assays for biological sensing.
Assuntos
Técnicas Biossensoriais/métodos , DNA Circular/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Aptâmeros de Nucleotídeos/química , Fagos Bacilares/enzimologia , Sequência de Bases , DNA Circular/metabolismo , DNA Polimerase Dirigida por DNA/metabolismoRESUMO
Certain DNA polymerases, such as Ï29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super-long single-stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand-displacement ability of these polymerases. In this work, the ability of Ï29DNAP to carry out RCA over circular templates containing a protein-binding DNA aptamer sequence was investigated. It was found that protein-aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein-binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.
Assuntos
Aptâmeros de Nucleotídeos/biossíntese , Aptâmeros de Nucleotídeos/química , Proteínas de Ligação a DNA/química , DNA Polimerase Dirigida por DNA/química , Oligonucleotídeos/biossíntese , Oligonucleotídeos/química , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
Microbial pathogens pose serious threats to public health and safety, and results in millions of illnesses and deaths as well as huge economic losses annually. Laborious and expensive pathogen tests often represent a significant hindrance to implementing effective front-line preventative care, particularly in resource-limited regions. Thus, there is a significant need to develop low-cost and easy-to-use methods for pathogen detection. Herein, we present a simple and inexpensive litmus test for bacterial detection. The method takes advantage of a bacteria-specific RNA-cleaving DNAzyme probe as the molecular recognition element and the ability of urease to hydrolyze urea and elevate the pHâ value of the test solution. By coupling urease to the DNAzyme on magnetic beads, the detection of bacteria is translated into a pH increase, which can be readily detected using a litmus dye or pH paper. The simplicity, low cost, and broad adaptability make this litmus test attractive for field applications, particularly in the developing world.
Assuntos
Bactérias/genética , Bactérias/isolamento & purificação , Técnicas de Tipagem Bacteriana/métodos , DNA Catalítico/metabolismo , RNA Bacteriano/metabolismo , Corantes/análise , Corantes/química , DNA Catalítico/genética , Concentração de Íons de Hidrogênio , Papel , RNA Bacteriano/análise , RNA Bacteriano/genética , Especificidade da Espécie , Urease/metabolismoRESUMO
Saliva is an attractive sample for coronavirus disease 2019 testing due its ease of collection and amenability to detect viral RNA with minimal processing. Using a direct-to-RT-PCR method with saliva self-collected from confirmed COVID-19 positive volunteers, we observed 32% false negative results. Confirmed negative and healthy volunteer samples spiked with 106 genome copies/mL of heat-inactivated severe acute respiratory syndrome coronavirus 2 showed false negative results of 10% and 13%, respectively. Additional sample heating or dilution of the false negative samples conferred only modest improvements. These results highlight the potential to significantly underdiagnose COVID-19 infections when testing directly from minimally processed heterogeneous saliva samples.
Assuntos
Teste de Ácido Nucleico para COVID-19 , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Reações Falso-Negativas , Voluntários Saudáveis , Humanos , Testes ImediatosRESUMO
We have made an accidental discovery of an unusual, single-stranded, guanine-rich DNA molecule that is capable of adopting a folded structure in 7 M urea (7MU) known to denature nucleic acid structures. The folding of this molecule requires Na+ and Mg2+ and the folded structure remains stable when subjected to denaturing (7MU) polyacrylamide gel electrophoresis. Results from sequence mutagenesis, DNA methylation, and circular dichroism spectroscopy studies suggest that this molecule adopts an intramolecular guanine-quadruplex structure with 5 layers of guanine tetrads. Our finding indicates that DNA has the ability to create extremely stable structural folds despite its limited chemical repertoire, making it possible to develop DNA-based systems for unconventional applications.