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1.
Angew Chem Int Ed Engl ; 60(45): 24266-24274, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34464491

RESUMO

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 480 pM, respectively, and binds pseudotyped lentiviruses expressing the wildtype and alpha trimeric spike proteins with affinity constants of 2.1 pM and 2.3 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 10 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/química
2.
Nucleic Acids Res ; 49(13): 7267-7279, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34232998

RESUMO

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 Aptâmeros
3.
Angew Chem Int Ed Engl ; 59(34): 14584-14592, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32470152

RESUMO

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 Atômica
4.
Chembiochem ; 21(14): 2029-2036, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32180322

RESUMO

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 in 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 11 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/genética
5.
Angew Chem Int Ed Engl ; 57(31): 9739-9743, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-29901854

RESUMO

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 in 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 in situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence in 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 Óptica
6.
Sci Rep ; 8(1): 1935, 2018 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-29386529

RESUMO

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.


Assuntos
Quadruplex G/efeitos dos fármacos , Ureia/farmacologia , Sequência de Bases , Íons , Metais/farmacologia , Fatores de Tempo
7.
Sci Rep ; 7(1): 3110, 2017 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-28596600

RESUMO

We report on a new colorimetric DNA detection method that takes advantage of the power of polymerase chain reaction (PCR) and the simplicity of the classic litmus test. The strategy makes use of a modified set of primers for PCR to facilitate ensuing manipulations of resultant DNA amplicons: their tagging with urease and immobilization onto magnetic beads. The amplicon/urease-laden beads are then used to hydrolyze urea, resulting in the increase of pH that can be conveniently reported by a pH-sensitive dye. We have successfully applied this strategy for the detection of two hypervirulent strains of the bacterium Clostridium difficile that are responsible for the recent increase in the global incidence and severity of C. difficile infections. Furthermore, the viability of this test for diagnostic applications is demonstrated using clinically validated stool samples from C. difficile infected patients.


Assuntos
Colorimetria/métodos , Replicação do DNA , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos
8.
J Vis Exp ; (115)2016 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-27685457

RESUMO

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.


Assuntos
Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Colorimetria/métodos , Amônia/análise , Bactérias/enzimologia , DNA Catalítico/química , Enzimas Imobilizadas/química , Concentração de Íons de Hidrogênio , Sistemas Automatizados de Assistência Junto ao Leito , Urease/química
9.
Chembiochem ; 17(12): 1142-5, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-26994736

RESUMO

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 Nucleico
10.
Angew Chem Int Ed Engl ; 55(7): 2431-4, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26676768

RESUMO

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 in 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 in 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/metabolismo
11.
Chemistry ; 21(22): 8069-74, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25877998

RESUMO

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/metabolismo
12.
Angew Chem Int Ed Engl ; 53(47): 12799-802, 2014 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-25213464

RESUMO

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 pH 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/metabolismo
13.
Chemistry ; 20(9): 2420-4, 2014 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-24590539

RESUMO

Certain DNA polymerases, such as ϕ29 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 Ï•29DNAP 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/métodos
14.
Biomolecules ; 3(3): 563-77, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-24970181

RESUMO

Bacterial detection plays an important role in protecting public health and safety, and thus, substantial research efforts have been directed at developing bacterial sensing methods that are sensitive, specific, inexpensive, and easy to use. We have recently reported a novel "mix-and-read" assay where a fluorogenic DNAzyme probe was used to detect model bacterium E. coli. In this work, we carried out a series of optimization experiments in order to improve the performance of this assay. The optimized assay can achieve a detection limit of 1000 colony-forming units (CFU) without a culturing step and is able to detect 1 CFU following as short as 4 h of bacterial culturing in a growth medium. Overall, our effort has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that employs a catalytic DNA.

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