Duplex-specific Nuclease Signal Amplification Research Articles

Simple and Sensitive Quantification of MicroRNAs via PS@Au Microspheres-Based DNA Probes and DSN-Assisted Signal Amplification Platform.

Identifying the microRNA (miRNA) expression level can provide critical information for early diagnosis of cancers or monitoring the cancer therapeutic efficacy. This paper focused on a kind of gold-nanoparticle-coated polystyrene microbeads (PS@Au microspheres)-based DNA probe as miRNA capture and duplex-specific nuclease (DSN) signal amplification platform based on an RGB value readout for detection of miRNAs. In virtue of the outstanding selectivity and simple experimental operation, 5'-fluorochrome-labeled molecular beacons (MBs) were immobilized on PS@Au microspheres via their 3'-thiol, in the wake of the fluorescence quenching by nanoparticle surface energy transfer (NSET). Target miRNAs were captured by the PS@Au microspheres-based DNA probe through DNA/RNA hybridization. DSN enzyme subsequently selectively cleaved the DNA to recycle the target miRNA and release of fluorophores, thereby triggering the signal amplification with more free fluorophores. The RGB value measurement enabled a detection limit of 50 fM, almost 4 orders of magnitude lower than PS@Au microspheres-based DNA probe detection without DSN. Meanwhile, by different encoding of dyes, miRNA-21 and miRNA-10b were simultaneously detected in the same sample. Considering the ability for quantitation, high sensitivity, and convenient merits, the PS@Au microspheres-based DNA probe and DSN signal amplification platform supplied valuable information for early diagnosis of cancers.

Switchable Target-Responsive 3D DNA Hydrogels As a Signal Amplification Strategy Combining with SERS Technique for Ultrasensitive Detection of miRNA 155.

Usually, SERS technology requires labeling of the Raman reporter to obtain characteristic spectra for detection of biological samples. However, the number of labeled Raman reporters is often limited, resulting in the restricted improvement for sensitivity of SERS biosensor. In this work, switchable target-responsive 3D DNA hydrogels were introduced to precisely control trapping and release of Raman reporter toluidine blue (TB), which not only avoid labeling signal molecule but also improve the sensitivity of miRNA detection due to immobilization of abundant TB. In the absence of target miRNA, the DNA hydrogel presented a weak Raman signal because TB was far away from SERS substrates with an "OFF" status. However, the DNA hydrogel can be opened by the target miRNA to release TB producing strong Raman signal with an "ON" status. On the basis of this sensitive strategy, this switchable DNA hydrogel-based SERS platform can achieve quantitative detection of miRNA 155. Simultaneously, flexible SERS substrate (leaf@nafion@Ag) and target miRNA-induced duplex-specific nuclease signal amplification strategy were employed to significantly improve the sensitivity of the SERS platform. As a result, the as-proposed SERS platform can sensitively and selectively detect miRNA 155 with a wide linear range of 0.1 fM to 100 pM and low detection limit of 0.083 fM, which indicated that the platform has great potential to be applied in miRNA-related clinical diagnostics and biochemical researches.

Double-loop hairpin probe and doxorubicin-loaded gold nanoparticles for the ultrasensitive electrochemical sensing of microRNA

An electrochemical microRNA (miRNA) analysis platform by combining double-loop hairpin probe (DHP) and doxorubicin-loaded gold nanoparticles (AuNPs@Dox) for ultrasensitive miRNA detection is proposed. Firstly, we here report a DHP that is simultaneously engineered to incorporate a miRNA recognition sequence, an output segment and output's complementary fragment. The important aspect of this hairpin probe is that it would not be degraded by duplex specific nuclease (DSN) and circumvents elaborately chemical modification disadvantages encountered by classic molecular beacon. For the DHP-based DSN signal amplification system, DHP hybridizes with target miRNA to form DNA-miRNA heteroduplexes, and the DSN can hydrolyze the DNA in the heteroduplexes structure selectively, while released target miRNA strand can initiate another cycle resulting in a significant signal amplification and the accumulated output segments could be responsible for strand displacement on the electrode directly. Furthermore, a great deal of doxorubicin (Dox) are loaded on the gold nanoparticles (AuNPs) to fabricate the AuNPs@Dox biocomposites that could magnify the electrochemical signal and enable the ultrasensitive analysis of miRNA. As a result, the miRNA was capable of being detected in a limit of 0.17pM and other kinds of miRNA were discriminated facilely by this method. The described DHP as a toolbox and the nano-biocomposites as a novel signal material would not only promote the design of electrochemical biosensors but also open a good way to promote the establishment of test method in malignant tumors.

A gold nanoparticle-based colorimetric strategy coupled to duplex-specific nuclease signal amplification for the determination of microRNA

The authors report on a new gold nanoparticle-based colorimetric assay for microRNA (miRNA). It is based on duplex-specific nuclease (DSN)-assisted signal amplification. Following hybridization between target miRNA and its complementary single-stranded DNA (ssDNA), the ssDNA in the DNA:RNA hybrid is selectively cleaved by DSN to produce small DNA fragments. Target microRNA is thus released and will initiate another round of hybridization and DSN digestion. In this manner, each miRNA target can specifically trigger several cycles of hybridization and DSN cleavages to yield numerous small fragments of DNA oligonucleotides. The short DNA fragments (unlike the uncleaved ssDNA probe) exhibit superior capability for stabilizing unmodified AuNP against salt-induced aggregation to the uncleaved ssDNA probe, with an accompanying color change from blue to red. These findings are exploited in a colorimetric assay for miRNA that has a 10 fmol detection limit.

Sensitive detection of microRNA in complex biological samples by using two stages DSN-assisted target recycling signal amplification method

MicroRNA (miRNA) has become an important biomarker candidate for cancer diagnosis, prognosis, and therapy. In this study, we have developed a novel fluorescence method for sensitive and specific miRNA detection via duplex specific nuclease (DSN) signal amplification and demonstrated its practical application in biological samples. Malachite green (MG) was employed as a “label-free" signal transducer since fluorescence of MG could be enhanced by 100-fold when MG were binding to a G-quadruplex structure formed within the d(G2T)13G sequence. The proposed signal amplification strategy is an integrated “biological circuit” designed to initiate a cascade of enzymatic reactions in order to detect, amplify, and measure a specific miRNA sequence by using the isothermal cleavage property of a DSN. The circuit is composed of two molecular switches operating in series: the amplification reaction activated by a specific miRNA and the strand-displacement polymerization reaction designed to initiate molecular beacon-assisted amplification and signal transduction by using MG/G-quadruplex complex. The hsa-miR-141 (miR141) was chosen as a target miRNA because its level specifically abnormal in a wide range of common human cancers including breast, lung, colon, and prostate cancer. The proposed method allowed quantitative sequence-specific detection of miR141 (with a detection limit of 1.03pM) in a dynamic range from 1pM to 10μM, with an excellent ability to discriminate differences in miRNAs. Moreover, the detection assay was applied to quantify miR141 in cancerous cell lysates. On the basis of these findings, we believe that this proposed sensitive and specific assay has great potential as a miRNA quantification method for use in biomedical research and clinical diagnosis.

A new method for sensitive detection of microphthalmia-associated transcription factor based on “OFF-state” and “ON-state” equilibrium of a well-designed probe and duplex-specific nuclease signal amplification

Herein, we report a new method which the “stage change” of DNA1 and the cleavage feature upon recycled recognition by DSN was used to detect target microphthalmia-associated transcription factor (MITF) in cell nuclear extracts. In this method, we employed a well-designed DNA1 as a recognition element which can converting in two states, “ON-state” and “OFF-state”. Also, the DNA1 is modified with 2-OMe-RNA on hybridization part in the “OFF-state” to prevent meaningless digestion. By taking advantage of the high amplification efficiency of DSN-aided recycling, high sensitivity of MITF is realized with a detection limit as low as 1.1 pM, which is superior or comparable to that of the reported literature. This method is a fast and easy-to-use one-pot method that was carried out in a tube, while being quantitative and applicable to other proteins in the sample without involving complicated procedures or sophisticated instrumentations. It is very simple and fast, needing only mixing of DNA1, DNA2, MITF and DSN enzyme and incubating within 60min, which is in the homogeneous solution, and not requiring separation and troublesome procedures. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method holds great promise of becoming a routine tool for simultaneously quantitative analysis of multiple proteins and supplies valuable information for transcription factor-based early stage cancer diagnosis.

Fe3O4@Ag magnetic nanoparticles for microRNA capture and duplex-specific nuclease signal amplification based SERS detection in cancer cells

A functionalized Fe3O4@Ag magnetic nanoparticle (NP) biosensor for microRNA (miRNA) capture and ultrasensitive detection in total RNA extract from cancer cells was reported in this paper. Herein, Raman tags-DNA probes modified Fe3O4@Ag NPs were designed both as surface-enhanced Raman scattering (SERS) SERS and duplex-specific nuclease signal amplification (DSNSA) platform. Firstly, target miRNAs were captured to the surface of Fe3O4@Ag NPs through DNA/RNA hybridization. In the presence of endonuclease duplex specific nuclease (DSN), one target miRNA molecule could rehybrid thousands of DNA probes to trigger the signal-amplifying recycling. Base on the superparamagnetic of Fe3O4@Ag NPs, target miRNA let-7b can be captured, concentrated and direct quantified within a PE tube without any PCR preamplification treatment. The detection limit was 0.3fM (15 zeptomole, 50μL), nearly 3 orders of magnitude lower than conventional fluorescence based DSN biosensors for miRNA(∼100fM), even single-base difference between the let-7 family members can be discriminated. The result provides a novel proposal to combine the perfect single-base recognition and signal-amplifying ability of the endonuclease DSN with cost-effective SERS strategy for miRNA point-of-care (POC) clinical diagnostics.

Integration of DNA bio-gates and duplex-specific nuclease signal amplification: towards electrochemiluminescence detection of survivin mRNA.

Using the DNA bio-gate and duplex-specific nuclease assisted target recycling, a facile electrochemiluminescence assay was developed for the sensitive detection of survivin mRNA.

Highly Sensitive and Selective Strategy for MicroRNA Detection Based on WS2 Nanosheet Mediated Fluorescence Quenching and Duplex-Specific Nuclease Signal Amplification

MicroRNAs (miRNAs) play vital roles in physiologic and pathologic processes and are significant biomarkers for disease diagnostics and therapeutics. However, rapid, low-cost, sensitive, and selective detection of miRNAs remains a challenge because of their short length, sequence homology, and low abundance. Herein, we report for the first time that WS2 nanosheet can exhibit differential affinity toward short oligonucleotide fragment versus ssDNA probe and act as an efficient quencher for adsorbed fluorescent probes. This finding is utilized to develop a new strategy for simple, sensitive, and selective detection of miRNA by combining WS2 nanosheet based fluorescence quenching with duplex-specific nuclease signal amplification (DSNSA). This assay exhibits highly sensitive and selective with a detection limit of 300 fM and even discriminate single-base difference between the miRNA family members. The result indicates that this simple and cost-effective strategy holds great potential application in biomedical research and clinical diagnostics.

Backbone-modified molecular beacons for highly sensitive and selective detection of microRNAs based on duplex specific nuclease signal amplification

Based on backbone-modified molecular beacons and duplex-specific nuclease, we have developed a target recycling amplification method for highly sensitive and selective miRNA detection. The combination of a low fluorescence background of 2-OMe-RNA modified MB and nuclease-assisted signal amplification leads to ultrahigh assay sensitivity, and the powerful discriminating ability of MB enables the differentiation of highly similar miRNAs with one-base difference, both of which are of great significance to miRNA detection.

One-Step, Multiplexed Fluorescence Detection of microRNAs Based on Duplex-Specific Nuclease Signal Amplification

Traditional molecular beacons, widely applied for detection of nucleic acids, have an intrinsic limitation on sensitivity, as one target molecule converts only one beacon molecule to its fluorescent form. Herein, we take advantage of the duplex-specific nuclease (DSN) to create a new signal-amplifying mechanism, duplex-specific nuclease signal amplification (DSNSA), to increase the detection sensitivity of molecular beacons (Taqman probes). DSN nuclease is employed to recycle the process of target-assisted digestion of Taqman probes, thus, resulting in a significant fluorescence signal amplification through which one target molecule cleaves thousands of probe molecules. We further demonstrate the efficiency of this DSNSA strategy for rapid direct quantification of multiple miRNAs in biological samples. Our experimental results showed a quantitative measurement of sequence-specific miRNAs with the detection limit in the femtomolar range, nearly 5 orders of magnitude lower than that of conventional molecular beacons. This amplification strategy also demonstrated a high selectivity for discriminating differences between miRNA family members. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method promises a great potential of becoming a routine tool for simultaneously quantitative analysis of multiple miRNAs in tissues or cells, and supplies valuable information for biomedical research and clinical early diagnosis.