RESUMEN
Regulatory modules for controlling the kinetics of toehold-mediated strand displacement (TMSD) play critical roles in designing dynamic and dissipative DNA chemical reaction networks (CRNs) but are hardwired into sequence designs. Herein, we introduce antitoehold (At), a plug-and-play module for reversible and continuous tuning of TMSD kinetics by temporarily occupying the toehold domain via a metastable duplex and base stacking. We demonstrate that kinetic control can be readily activated or deactivated in real time for any TMSD by simply adding At or anti-At. Continuous tuning of TMSD kinetics can also be achieved by altering the concentration of At. Moreover, the simple addition of At could readily reprogram existing TMSDs into a pulse-generation DNA CRN with continuous tunability. Our At approach also offers a new way for engineering continuously tunable DNA hybridization probes, which may find practical uses for discriminating clinically important mutations. Because of the simplicity, we anticipate that At will find wide applications for engineering DNA CRNs with diverse dynamic and dissipative behaviors, and DNA hybridization probes with tunable affinity and selectivity.
Asunto(s)
ADN , ADN/química , Hibridación de Ácido Nucleico , Cinética , Sondas de ADNRESUMEN
Somatic mutations are important signatures in clinical cancer treatment. However, accurate detection of rare somatic mutations with low variant-allele frequencies (VAFs) in clinical samples is challenging because of the interference caused by high concentrations of wild-type (WT) sequences. Here, we report a post amplification SNV-specific DNA assembly (PANDA) technology that eliminates the high concentration pressure caused by WT through a mismatch-guided DNA assembly and enables the ultrasensitive detection of cancer mutations with VAFs as low as 0.1%. Because it generates an assembly product that only exposes a single-stranded domain with the minimal length for signal readout and thus eliminates possible interferences from secondary structures and cross-interactions among sequences, PANDA is highly versatile and expandable for multiplex testing. With ultrahigh sensitivity, PANDA enabled the quantitative analysis of EGFR mutations in cell-free DNA of 68 clinical plasma samples and four pleuroperitoneal fluid samples, with test results highly consistent with NGS deep sequencing. Compared to digital PCR, PANDA returned fewer false negatives and ambiguous cases of clinical tests. Meanwhile, it also offers much lower upfront instrumental and operational costs. The multiplexity was demonstrated by developing a 3-plex PANDA for the simultaneous analysis of three EGFR mutations in 54 pairs of tumor and the adjacent noncancerous tissue samples collected from lung cancer patients. Because of the ultrahigh sensitivity, multiplexity, and simplicity, we anticipate that PANDA will find wide applications for analyzing clinically important rare mutations in diverse devastating diseases.
Asunto(s)
Ácidos Nucleicos Libres de Células , Neoplasias Pulmonares , Humanos , Alelos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , ADN/genética , Receptores ErbBRESUMEN
Synthetic DNA walkers that are inspired by the walking behaviors of naturally occurring motor proteins have emerged into an important subfield of DNA nanotechnology. While early DNA walkers were designed to walk on one-dimensional (1D) DNA tracks, the development of DNA origami and DNA functionalized micro-/nanomaterials has enabled diverse 2D and 3D tracks. Random walking becomes possible in such platforms and such stochastic DNA walkers can be engineered with much-improved speed and processivity. The invention and improvement of diverse stochastic DNA walkers have made them ideal amplification platforms for analytical and diagnostic applications. In this feature article, we first review the development of DNA walkers with a historical aspect and then focus on the advances in stochastic DNA walkers. We finally elaborated our research efforts to design varying 3D stochastic DNA walkers for rapid and amplified detection of biologically important nucleic acids and proteins.
Asunto(s)
ADN , Nanoestructuras , ADN/metabolismo , Nanotecnología/métodosRESUMEN
Combining experimental and simulation strategies to facilitate the design and operation of nucleic acid hybridization probes are highly important to both fundamental DNA nanotechnology and diverse biological/biomedical applications. Herein, we introduce a DNA equalizer gate (DEG) approach, a class of simulation-guided nucleic acid hybridization probes that drastically expand detection windows for discriminating single nucleotide variants in double-stranded DNA (dsDNA) via the user-definable transformation of the quantitative relationship between the detection signal and target concentrations. A thermodynamic-driven theoretical model was also developed, which quantitatively simulates and predicts the performance of DEG. The effectiveness of DEG for expanding detection windows and improving sequence selectivity was demonstrated both in silico and experimentally. As DEG acts directly on dsDNA, it is readily adaptable to nucleic acid amplification techniques, such as polymerase chain reaction (PCR). The practical usefulness of DEG was demonstrated through the simultaneous detection of infections and the screening of drug-resistance in clinical parasitic worm samples collected from rural areas of Honduras.
Asunto(s)
Sondas de ADN/química , Colorantes Fluorescentes/química , Animales , ADN/química , Helmintos/genética , Helmintos/aislamiento & purificación , Modelos Teóricos , Hibridación de Ácido Nucleico/métodos , Nucleótidos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , TermodinámicaRESUMEN
There is a need for biosensing systems that can be operated at the point-of-care (POC) for disease screening and diagnostics and health monitoring. In spite of this, simple to operate systems with the required analytical sensitivity and specificity in clinical samples, using a sample-in-answer-out approach, remain elusive. Reported here is an electrochemical bio-barcode assay (e-biobarcode assay) that integrates biorecognition with signal transduction using molecular (DNA/protein) machines and signal readout using nanostructured electrodes. The e-biobarcode assay eliminates multistep processing and uses a single step for analysis following sample collection into the reagent tube. A clinically relevant performance for the analysis of prostate specific antigen (PSA) in undiluted and unprocessed human plasma: a log-linear range of 1â ng mL-1 -200â ng mL-1 and a LOD of 0.4â ng mL-1 , was achieved. The e-biobarcode assay offers a realistic solution for biomarker analysis at the POC.
Asunto(s)
Biomarcadores de Tumor/sangre , Técnicas Biosensibles , Técnicas Electroquímicas , Sistemas de Atención de Punto , Antígeno Prostático Específico/sangre , Electrodos , HumanosRESUMEN
Miniaturization of nucleic acid tests (NATs) into portable, inexpensive detection platforms may aid disease diagnosis in point-of-care (POC) settings. Colorimetric signals are ideal readouts for portable NATs, and it remains of high demand to develop color readouts that are simple, quantitative, and versatile. Thus motivated, we report a fast light-activated substrate chromogenic polymerase chain reaction (FLASH PCR) that uses DNA intercalating dyes (DIDs) to enable colorimetric nucleic acid detection and quantification. The FLASH system is established on our finding that DID-DNA intercalation can promote the rapid photooxidation of chromogenic substrates through light-induced production of singlet oxygen. Using this principle, we have successfully converted DID-based fluorescent PCR assays into colorimetric FLASH PCR. To demonstrate the practical applicability of FLASH PCR to POC diagnosis, we also fabricated two readout platforms, including a portable electronic FLASH reader and a paper-based FLASH strip. Using the FLASH reader, we were able to detect as low as 60 copies of DNA standards, a limit of detection (LOD) comparable with commercial quantitative PCR. The FLASH strip further enables the reader-free detection of PCR amplicons by converting the colorimetric signal into the visual measurement of distance as a readout. Finally, the practical applicability of the FLASH PCR was demonstrated by the detection and/or quantification of nucleic acid markers in diverse clinical and biological samples.
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Compuestos Cromogénicos/análisis , Colorimetría , ADN/análisis , ADN/genética , Luz , Reacción en Cadena de la PolimerasaRESUMEN
Single-nucleotide variants (SNVs) that are strongly associated with many genetic diseases and tumors are important both biologically and clinically. Detection of SNVs holds great potential for disease diagnosis and prognosis. Recent advances in DNA nanotechnology have offered numerous principles and strategies amenable to the detection and quantification of SNVs with high sensitivity, specificity, and programmability. In this review, we will focus our discussion on emerging techniques making use of DNA strand displacement, a basic building block in dynamic DNA nanotechnology. Based on their operation principles, we classify current SNV detection methods into three main categories, including strategies using toehold-mediated strand displacement reactions, toehold-exchange reactions, and enzyme-mediated strand displacement reactions. These detection methods discriminate SNVs from their wild-type counterparts through subtle differences in thermodynamics, kinetics, or response to enzymatic manipulation. The remarkable programmability of dynamic DNA nanotechnology also allows the predictable design and flexible operation of diverse strand displacement probes and/or primers. Here, we offer a systematic survey of current strategies, with an emphasis on the molecular mechanisms and their applicability to in vitro diagnostics.
Asunto(s)
ADN/química , ADN/genética , Variación Genética , Nucleótidos/genética , Humanos , Nanotecnología , Hibridación de Ácido Nucleico , Sondas de Ácido Nucleico/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
Herein, we report a bottom-up approach to assemble a series of stochastic DNA walkers capable of probing dynamic interactions occurring at the bio-nano interface. We systematically investigated the impact of varying interfacial factors, including intramolecular interactions, orientation, cooperativity, steric effect, multivalence, and binding hindrance on enzymatic behaviors at the interfaces of spherical nucleic acids. Our mechanistic study has revealed critical roles of various interfacial factors that significantly alter molecular binding and enzymatic behaviors from bulk solutions. The improved understanding of the bio-nano interface may facilitate better design and operation of nanoparticle-based biosensors and/or functional devices. We successfully demonstrate how improved understanding of the bio-nano interface help rationalize the design of amplifiable biosensors for nucleic acids and antibodies.
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ADN/química , Oro/química , Nanopartículas del Metal/química , Técnicas Biosensibles , Hibridación de Ácido Nucleico , Tamaño de la Partícula , Procesos Estocásticos , Propiedades de SuperficieRESUMEN
Rapid, low-cost, and sensitive nucleic acid detection and quantification assays enabled by microfluidic paper-based analytical devices (µPADs) hold great promise for point-of-care disease diagnostics and field-based molecular tests. Through the capillary action in µPAD, flexible manipulation of nucleic acid samples can be achieved without the need for external pumps or power supplies, making it possible to fabricate highly integrated sample-to-answer devices that streamline the nucleic acid extraction, separation, concentration, amplification, and detection. To detect minute amounts of genetic materials from clinical and biological samples, it is also critical to develop sensitive signal readouts that generate physically detectable signals for in-device nucleic acid detection and/or quantification. In this review, we will focus on µPAD approaches for the facile manipulation of nucleic acids and emerging signal transduction strategies allowing sensitive and specific nucleic acid detection in µPAD. Graphical abstract á .
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Ácidos Nucleicos/análisis , Papel , Colorantes/química , Hibridación de Ácido Nucleico , Sistemas de Atención de Punto , Transducción de Señal , Biología SintéticaRESUMEN
Single nucleotide variants (SNVs) are important both clinically and biologically because of their profound biological consequences. Herein, we engineered a nicking endonuclease-powered three dimensional (3D) DNA nanomachine for discriminating SNVs with high sensitivity and specificity. Particularly, we performed a simulation-guided tuning of sequence designs to achieve the optimal trade-off between device efficiency and specificity. We also introduced an auxiliary probe, a molecular fuel capable of tuning the device in solution via noncovalent catalysis. Collectively, our device produced discrimination factors comparable with commonly used molecular probes but improved the assay sensitivity by â¼100 times. Our results also demonstrate that rationally designed DNA probes through computer simulation can be used to quantitatively improve the design and operation of complexed molecular devices and sensors.