Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A sensitive and specific method for microRNA detection and in situ imaging based on a CRISPR-Cas9 modified catalytic hairpin assembly.

ABSTRACT: We report here a method for the molecular detection of miRNAs in exosomes and imaging in living cells based on CRISPR-Cas9 and catalytic hairpin assembly. With the cascade signal amplification of catalytic hairpin assembly and high specific recognition of CRISPR-Cas9, the detection method showed a sensitivity of 23.5 fM and was applied for accurate exosomal miR-21 detection and in situ miR-21 imaging.

SUBMITTER: Liu Y

PROVIDER: S-EPMC9055656 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A sensitive and specific method for microRNA detection and <i>in situ</i> imaging based on a CRISPR-Cas9 modified catalytic hairpin assembly.

Liu Yang Y Li Shihong S Zhang Likun L Zhao Qian Q Li Nuo N Wu Yuxin Y

RSC advances 20200728 47

We report here a method for the molecular detection of miRNAs in exosomes and imaging in living cells based on CRISPR-Cas9 and catalytic hairpin assembly. With the cascade signal amplification of catalytic hairpin assembly and high specific recognition of CRISPR-Cas9, the detection method showed a sensitivity of 23.5 fM and was applied for accurate exosomal miR-21 detection and <i>in situ</i> miR-21 imaging. ...[more]

PMID: 35519131

Similar Datasets

Catalytic hairpin assembly gel assay for multiple and sensitive microRNA detection.

Project description:As important modulators of gene expression, microRNAs (miRNAs) have been identified as promising biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. Here we report a facile, multiple and sensitive miRNA detection method that uses conventional gel electrophoresis and catalytic hairpin assembly (CHA) system without any complex nanomaterials or enzymatic amplification. Methods: In this study, three pairs of hairpin probes are rationally designed with thermodynamically and kinetically preferable feasibility for the CHA process. In the present of target miRNA, the stem of the corresponding hairpin detection probe (HDP) will be unfolded and expose the concealed domain. The corresponding hairpin assistant probe (HAP) then replaces the hybridized target miRNA to form specific HDP/HAP complexes and releases miRNA based on thermodynamically driven entropy gain process, and the released miRNA triggers the next recycle to produce tremendous corresponding HDP/HAP complexes. Results: The results showed that the CHA gel assay can detect miRNA at fM levels and shows good capability of discriminating miRNA family members and base-mismatched miRNAs. It is able to analyze miRNAs extracted from cell lysates, which are consistent with the results of conventional polymerase chain reaction (PCR) method. Depending on the length of the designed hairpin probes, the CHA gel assay consisting of different hairpin probes effectively discriminated and simultaneously detected multiple miRNAs in homogenous solution and miRNAs extracted from cell lysates. Conclusion: The work highlights the practical use of a conventional gel electrophoresis for sensitive interesting nucleic acid sequences detection.

| S-EPMC5956999 | biostudies-literature

Circle Padlock-Mediated Catalytic Hairpin Assembly Cooperating Primer Exchange Reaction for Sensitive and Label-Free MicroRNA Detection

Project description: Not available

| S-EPMC11696746 | biostudies-literature

Genetically Encoded Catalytic Hairpin Assembly for Sensitive RNA Imaging in Live Cells.

Project description:DNA and RNA nanotechnology has been used for the development of dynamic molecular devices. In particular, programmable enzyme-free nucleic acid circuits, such as catalytic hairpin assembly, have been demonstrated as useful tools for bioanalysis and to scale up system complexity to an extent beyond current cellular genetic circuits. However, the intracellular functions of most synthetic nucleic acid circuits have been hindered by challenges in the biological delivery and degradation. On the other hand, genetically encoded and transcribed RNA circuits emerge as alternative powerful tools for long-term embedded cellular analysis and regulation. Herein, we reported a genetically encoded RNA-based catalytic hairpin assembly circuit for sensitive RNA imaging inside living cells. The split version of Broccoli, a fluorogenic RNA aptamer, was used as the reporter. One target RNA can catalytically trigger the fluorescence from tens-to-hundreds of Broccoli. As a result, target RNAs can be sensitively detected. We have further engineered our circuit to allow easy programming to image various target RNA sequences. This design principle opens the arena for developing a large variety of genetically encoded RNA circuits for cellular applications.

| S-EPMC6201751 | biostudies-literature

Chaperone Copolymer-Assisted Catalytic Hairpin Assembly for Highly Sensitive Detection of Adenosine.

Project description:Adenosine is an endogenous molecule that plays a vital role in biological processes. Research indicates that abnormal adenosine levels are associated with a range of diseases. The development of sensors capable of detecting adenosine is pivotal for early diagnosis of disease. For example, elevated adenosine levels are closely associated with the onset and progression of cancer. In this study, we designed a novel DNA biosensor utilizing chaperone copolymer-assisted catalytic hairpin assembly for highly sensitive detection of adenosine. The functional probe comprises streptavidin magnetic beads, an aptamer, and a catalytic chain. In the presence of adenosine, it selectively binds to the aptamer, displacing the catalytic chain into the solution. The cyclic portion of H1 hybridizes with the catalytic strand, while H2 hybridizes with the exposed H1 fragment to form an H1/H2 complex containing a G-quadruplex. Thioflavin T binds specifically to the G-quadruplex, generating a fluorescent signal. As a nucleic acid chaperone, PLL-g-Dex expedites the strand exchange reaction, enhancing the efficiency of catalytic hairpin assembly, thus amplifying the signal and reducing detection time. The optimal detection conditions were determined to be a temperature of 25 °C and a reaction time of 10 min. Demonstrating remarkable sensitivity and selectivity, the sensor achieved a lowest limit of detection of 9.82 nM. Furthermore, it exhibited resilience to interference in complex environments such as serum, presenting an effective approach for rapid and sensitive adenosine detection.

| S-EPMC11314456 | biostudies-literature

Catalytic Hairpin Assembly-Assisted Rolling Circle Amplification for High-Sensitive Telomerase Activity Detection.

Project description:Telomerase is a promising biomarker and a potential therapeutic target of malignant tumors. Reliable, facile, and sensitive telomerase activity analysis is of vital importance for both early diagnosis and therapy of malignant tumors. Herein, we proposed a novel fluorescent assay termed catalytic hairpin assembly-assisted rolling circle amplification (CAR) for both in vitro and in situ high-sensitive telomerase activity detection. In the presence of active telomerase, the extension of a designed telomerase primer was limited to five bases (GGGTT), thus forming short telomerase products. Afterward, the obtained telomerase extension products cyclized Padlock and subsequently initiated the rolling circle amplification (RCA). In order to maintain a higher sensitivity, an ingeniously designed catalytic hairpin assembly (CHA) was attached for both signal amplification and result readout. The highlights of the CAR method were concluded as follows: (i) dual signal amplification from CHA and RCA ensures high sensitivity and (ii) the CAR method has the potential for both in vitro and intracellular imaging of telomerase activity. We believe that the CAR method would be of great potential for the diagnosis and therapy of various human diseases.

| S-EPMC7254775 | biostudies-literature

A CRISPR/Cas9-mediated in situ complementation method for Phytophthora sojae mutants.

Project description:Phytophthora sojae is an important model species for oomycete functional genomics research. Recently, a CRISPR/Cas9-mediated genome-editing technology has been successfully established in P. sojae, which has been rapidly and widely applied in oomycete research. However, there is an emerging consensus in the biological community that a complete functional gene research system is needed such as developed in the investigations in functional complementation carried out in this study. We report the development of an in situ complementation method for accurate restoration of the mutated gene. We targeted a regulatory B-subunit of protein phosphatase 2A (PsPP2Ab1) to verify this knockout and subsequent complementation system. We found that the deletion of PsPP2Ab1 in P. sojae leads to severe defects in vegetative hyphal growth, soybean infection, and loss of the ability to produce sporangia. Subsequently, the reintroduction of PsPP2Ab1 into the knockout mutant remedied all of the deficiencies. This study demonstrates the successful implementation of an in situ complementation system by CRISPR/Cas9, which will greatly accelerate functional genomics research of oomycetes in the post-genomic era.

| S-EPMC7865083 | biostudies-literature

A DNA nanowire based localized catalytic hairpin assembly reaction for microRNA imaging in live cells.

Project description:Here, we have developed a localized catalytic hairpin assembly (LCHA) strategy for intracellular miR-21 imaging by using DNA nanowires confining both hairpin probes in a compact space. The LCHA is constructed by interval hybridization of DNA hairpin probe pairs to a DNA nanowire with multiplex footholds generated by alternating chain hybridization. Compared to the conventional catalytic hairpin assembly (CHA) strategy, the LCHA significantly shortens the reaction time and enhances the sensitivity. Moreover, the proposed LCHA can serve as a carrier for delivery of probes into live cells as well as protect the probes from nuclease degradation and enhances the stability. We anticipate that this design can be widely applied in facilitating basic biomedical research and disease diagnosis.

| S-EPMC6194499 | biostudies-literature

l-DNA-Based Catalytic Hairpin Assembly Circuit.

Project description:Isothermal, enzyme-free amplification methods based on DNA strand-displacement reactions show great promise for applications in biosensing and disease diagnostics but operating such systems within biological environments remains extremely challenging due to the susceptibility of DNA to nuclease degradation. Here, we report a catalytic hairpin assembly (CHA) circuit constructed from nuclease-resistant l-DNA that is capable of unimpeded signal amplification in the presence of 10% fetal bovine serum (FBS). The superior biostability of the l-DNA CHA circuit relative to its native d-DNA counterpart was clearly demonstrated through a direct comparison of the two systems (d versus l) under various conditions. Importantly, we show that the l-CHA circuit can be sequence-specifically interfaced with an endogenous d-nucleic acid biomarker via an achiral peptide nucleic acid (PNA) intermediary, enabling catalytic detection of the target in FBS. Overall, this work establishes a blueprint for the detection of low-abundance nucleic acids in harsh biological environments and provides further impetus for the construction of DNA nanotechnology using l-oligonucleotides.

| S-EPMC7070954 | biostudies-literature

CRISPR-Cas and catalytic hairpin assembly technology for target-initiated amplification detection of pancreatic cancer specific tsRNAs.

Project description:Transfer RNA-derived small RNAs (tsRNAs) tRF-LeuCAG-002 (ts3011a RNA) is a novel class of non-coding RNAs biomarker for pancreatic cancer (PC). Reverse transcription polymerase chain reaction (RT-qPCR) has been unfit for community hospitals that are short of specialized equipment or laboratory setups. It has not been reported whether isothermal technology can be used for detection, because the tsRNAs have rich modifications and secondary structures compared with other non-coding RNAs. Herein, we have employed a catalytic hairpin assembly (CHA) circuit and clustered regularly interspaced short palindromic repeats (CRISPR) to develop an isothermal and target-initiated amplification method for detecting ts3011a RNA. In the proposed assay, the presence of target tsRNA triggers the CHA circuit that transforms new DNA duplexes to activate collateral cleavage activity of CRISPR-associated proteins (CRISPR-Cas) 12a, achieving cascade signal amplification. This method showed a low detection limit of 88 aM at 37 °C within 2 h. Moreover, it was demonstrated for the first time that, this method is less likely to produce aerosol contamination than RT-qPCR by simulating aerosol leakage experiments. This method has good consistency with RT-qPCR in the detection of serum samples and showed great potential for PC-specific tsRNAs point-of-care testing (POCT).

| S-EPMC10188930 | biostudies-literature

A Dual-Cycle Isothermal Amplification Method for microRNA Detection: Combination of a Duplex-Specific Nuclease Enzyme-Driven DNA Walker with Improved Catalytic Hairpin Assembly.

Project description:The association between microRNAs and various diseases, especially cancer, has been established in recent years, indicating that miRNAs can potentially serve as biomarkers for these diseases. Determining miRNA concentrations in biological samples is crucial for disease diagnosis. Nevertheless, the stem-loop reverse transcription quantitative PCR method, the gold standard for detecting miRNA, has great challenges in terms of high costs and enzyme limitations when applied to clinical biological samples. In this study, an isothermal signal amplification method based on a duplex-specific nuclease (DSN) enzyme-driven DNA walker and an improved catalytic hairpin assembly (CHA) was designed for miRNA detection. First, biotin-triethylene glycol-modified trigger-releasable DNA probes were conjugated to the streptavidin-coated magnetic beads for recognizing the target miRNA. The DSN enzyme specifically hydrolyzes DNA strands when the DNA probe hybridizes with the targeted miRNA. This recycling process converts the input miRNA into short trigger fragments (catalysts). Finally, three hairpins of improved CHA are driven by this catalyst, resulting in the three-armed CHA products and a fluorescence signal as the output. This dual-cycle biosensor shows a good linear relationship in the detection of miR-21 and miR-141 over the final concentration range of 250 fM to 50 nM, presenting an excellent limit of detection (2.95 amol). This system was used to detect miR-21 and miR-141 in MCF-7 and 22RV1 cells, as well as in 1% human serum. This system can be used to evaluate the expression levels of miRNAs in different biological matrices for the clinical diagnosis and prognosis of different cancers.

| S-EPMC11766441 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data