Autonomous DNA Machine Research Articles

Trigger-activated autonomous DNA machine for amplified liver cancer biomarker microRNA21 imaging.

MicroRNA-21 (miRNA-21) is a kind of RNA that exists in biological fluids such as blood, urine and saliva. It has over expression in liver cancer and has different expression in different stages of cancer. However, due to the characteristics of small base number, short length, low abundance and easy degradation of miRNA-21, the detection of miRNA-21 is a challenging subject. Visualization, sensitive, specific and stable detection of tumor suppressor or oncogene microRNAs (miRNAs) remains challenging and is highly significant for clinical diagnostics. To solve this problem, we have developed a target-triggered hybridization assembly DNA machine for intracellular miRNA imaging based on strand displacement amplification (SDA) and branched hybridization chain reaction (B-HCR). In this approach, the target miRNA could hybridize with the template probe to trigger the SDA, resulting in the formation of nicked fragments (NFs) that hybridized with hairpin probe1 (HP1). The opened HP1 could hybridize with hairpin probe2 (HP2), leading to the self-assembly of hyperbranched DNA nanostructures through B-HCR. As expected, the newly developed method exhibits a detection limit down to 11.3pM miRNA-21 and achieves high selectivity toward miRNA-21 against other interfering miRNAs. Due to its superior sensitivity and selectivity, our method can be further used to detect miRNA-21 in human serum samples. By taking advantage of intelligent design, the proposed method was also used for image miRNA-21 expression levels in different cell lines. This method shows a broad application in clinical diagnosis.

A catalytic DNA circuit-programmed and enzyme-powered autonomous DNA machine for nucleic acid detection.

A catalytic DNA circuit was ingeniously engineered to harness the cleavage of a nicking endonuclease on a triple-stranded duplex DNA probe, autonomously actuating a successive enzyme-powered DNA machine for one-step, isothermal, and autocatalytic nucleic acid analysis. The detection performance could be tuned with or without the use of enzymes.

Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene

The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis.

In situ amplification signaling-based autonomous aptameric machine for the sensitive fluorescence detection ofcocaine

The development of autonomous DNA machines and their use for specific sensing purpose have recently attracted considerable research attention. In existing autonomous machines, the target recognition process and signal transduction are separated from each other. This results in misunderstanding of the operation behavior, and the assay capability is compromised when serving as a sensing tool. In this communication, the integrated signal transduction-based autonomous aptameric machine, in which the recognition element and signal reporters are integrated into a DNA strand, is developed. This new machine can execute the in situ amplification of target binding-induced signal. The authentic operation behavior of autonomous DNA machine is discovered: the machine's products directly hybridize to the “track” rather than to the signaling probes. Along this line, the machine is employed to detect the cocaine in a more straightforward fashion, and improved assay characteristics (for example, the dynamic response range is widened by more than 500-fold) are achieved. Our efforts not only clarify the concept described in traditional autonomous DNA machines but also have made technological advancements that are expected to be especially valuable in designing nucleic acid-based machines employed in basic research and medical diagnosis.

Dumbbell probe-mediated cascade isothermal amplification: A novel strategy for label-free detection of microRNAs and its application to real sample assay

Considering the great significance of microRNAs (miRNAs) in cancer detection and typing, the development of sensitive, specific, quantitative, and low-cost methods for the assay of expression levels of miRNAs is desirable. We describe a highly efficient amplification platform for ultrasensitive analysis of miRNA (taking let-7a miRNA as a model analyte) based on a dumbbell probe-mediated cascade isothermal amplification (DP-CIA) strategy. The method relies on the circularization of dumbbell probe by binding target miRNA, followed by rolling circle amplification (RCA) reaction and an autonomous DNA machine performed by nicking/polymerization/displacement cycles that continuously produces single-stranded G-quadruplex to assemble with hemin to generate a color signal. In terms of the high sensitivity (as low as 1zmol), wide dynamic range (covering 9 orders of magnitude), good specificity (even single-base difference) and easy operation (one probe and three enzymes), the proposed label-free assay is successfully applied to direct detection of let-7a miRNA in real sample (total RNA extracted from human lung tissue), demonstrating an attractive alternative for miRNA analysis for gene expression profiling and molecular diagnostics, particularly for early cancer diagnosis.

Surface-enhanced Raman scattering assay combined with autonomous DNA machine for detection of specific DNA and cancer cells

An ultrasensitive surface-enhanced Raman scattering (SERS) detection system based on DNA machine isothermal amplification is reported to detect a specific DNA sequence for the first time and was successfully applied to detect cancer cells.

Ultrasensitive Electrochemical Analysis of Two Analytes by Using an Autonomous DNA Machine that Works in a Two‐Cycle Mode

We report a novel autonomous DNA machine for amplified electrochemical analysis of two DNAs. The DNA machine operates in a two-cycle working mode to amplify DNA recognition events; the working mode is assisted by two different nicking endonucleases (NEases). Two bio-barcode probes, a ZnS nanoparticle (NP)-DNA probe and a CdS NP-DNA probe, were used to trace two target DNAs. The detection system was based on a sensitive differential pulse anodic stripping voltammetry (DPASV) method for the simultaneous detection of Zn(II) and Cd(II) tracers, which were obtained by dissolving the two probes. Under the optimised conditions, detection limits as low as 5.6×10(-17) (3σ) and 4.1×10(-17) M (3σ) for the two target DNAs were achieved. It has been proven that the DNA machine system can simultaneously amplify two target DNAs by more than four orders of magnitude within 30 min at room temperature. In addition, in combination with an aptamer recognition strategy, the DNA machine was further used in the aptamer-based amplification analysis of adenosine triphosphate (ATP) and lysozyme. With the amplification of the DNA machine, detection limits as low as 5.6×10(-9) M (3σ) for ATP and 5.2×10(-13) M (3σ) for lysozyme were simultaneously obtained. The satisfactory determination of ATP and lysozyme in Ramos cells reveals the good selectivity and feasibility of this protocol. The DNA machine is a promising tool for ultrasensitive and simultaneous multianalysis because of its remarkable signal amplification and simple machine-like operation.

Isothermal sensitive detection of microRNA using an autonomous DNA machine recycling output as input

An autonomous DNA machine recycling the output as the input for isothermal, sensitive, and specific detection of miRNAs has been developed. This machine shows considerably high signal amplification efficiency (∼1000-fold) and thus a low detection limit (∼20 amol). The machine also shows high specificity, discriminating 50 amol of synthetic miRNA from 100-fold larger amounts of its family member and from 100 ng of unrelated total RNAs. Moreover, it is available for practically detecting natural miRNAs in total RNAs.

Inhibition of the In Vitro Replication of DNA by an Aptamer-Protein Complex in an Autonomous DNA Machine

DNA replication plays a central role in living organisms. Unregulated or uncontrollable DNA replication is well known to result in many pathological states, such as cancer, autoimmune diseases, and viral/bacterial infections. We report that an aptamer-protein complex could indirectly inhibit in vitro replication of DNA. An isothermal DNA machine based on the strand-displacement amplification is employed to support our assumption. An antithrombin aptamer sequence is rationally encoded into the DNA replication template. Once thrombin binds to the template, the as-formed aptamer-protein complexes can, in turn, become a barrier to the polymerase and inhibit the DNA replication activities in both static and dynamic modes. The inhibition is successfully confirmed by both fluorescence and gel-electrophoresis experiments. Considering the availability of a broad library of aptamers and the existence of various DNA/protein interactions, our results imply the possibility for the rational regulation of DNA replication in vivo.

The Aggregation of Au Nanoparticles by an Autonomous DNA Machine Detects Viruses

The Aggregation of Au Nanoparticles by an Autonomous DNA Machine Detects Viruses

A Virus Spotlighted by an Autonomous DNA Machine

A Virus Spotlighted by an Autonomous DNA Machine

A Virus Spotlighted by an Autonomous DNA Machine

Hochempfindlich: Zur Analyse viraler DNA (M13-Phage) eignet sich eine DNA-basierte Maschine, die durch dNTPs angetrieben wird und DNAzym-Reportereinheiten synthetisiert. Die DNAzyme imitieren Peroxidase und führen zu einem Auslesen der analysierten DNA mithilfe von Kolorimetrie oder Chemilumineszenz.