Bis-enzyme cascade CRISPR-Cas12a platform for miRNA detection

DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong trans-cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the trans-cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the trans-cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.

Section: Resultsmentioning

confidence: 99%

CRISPR-Cas12a Coupled with DNA Nanosheet-Amplified Fluorescence Anisotropy for Sensitive Detection of Biomolecules

Xie

Luo

et al. 2023

“…For example, Yang et al described a rapid and sensitive detection method named BPTC by integrating T7 RNA polymerase, the phi29 polymerase with CRISPR-Cas12a, although it is unsatisfactory due to its additional reagents and multistep procedures. 30 Xie et al developed a stable sensing platform by combining reverse transcription with T7 RNA transcription, realizing the sensitive miRNA detection in clinical samples, which also requires multiple primers and multistep procedures. 31 Herein, we described a novel T7 RNA polymerase-based isothermal transcription amplification method for one-step sensitivity detection of microRNA by utilizing specially designed single-stranded DNA hairpin switches.…”

Section: ■ Introductionmentioning

confidence: 99%

“…26,27 It is widely used in research and commercial development due to its applicability for in vitro RNA production, its high yield, and the high fidelity of the transcripts produced. 28,29 However, in most T7 RNA polymerase-dependent amplification methods, T7 RNA polymerase-based transcription reactions are designed to be combined with other amplification steps in order to improve the sensitivity, 30,31 which inevitably requires multiple primers, additional reagents, and multistep procedures. Meanwhile, due to the linear primer design, many methods exhibited insufficient specificity.…”

Section: ■ Introductionmentioning

confidence: 99%

“…However, this thermal-cycling method is time-consuming, sometimes nonspecific, and limited to a laboratory setting, which would restrict the application in resource-limited locations. To circumvent this problem, various isothermal nucleic acid amplification techniques have been developed, such as rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), , strand displacement reaction (SDA) and catalytic hairpin assembly (CHA). , Recently, T7 in vitro RNA transcription amplification methods have found a growing interest in detecting nucleic acid targets with high amplification efficiency. ,− T7 RNA polymerase-based transcription amplification is a novel isothermal RNA amplification method that catalyzes the synthesis of the RNA fragment in a 5′–3′ direction after the recruitment of T7 RNA polymerase and a specific T7 promoter. , It is widely used in research and commercial development due to its applicability for in vitro RNA production, its high yield, and the high fidelity of the transcripts produced. , However, in most T7 RNA polymerase-dependent amplification methods, T7 RNA polymerase-based transcription reactions are designed to be combined with other amplification steps in order to improve the sensitivity, , which inevitably requires multiple primers, additional reagents, and multistep procedures. Meanwhile, due to the linear primer design, many methods exhibited insufficient specificity.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Hairpin Switches-Based Isothermal Transcription Amplification for Simple, Sensitivity Detection of MicroRNA

Long,

Luo,

Yuan

et al. 2023

The ability to simply, selectively, and sensitively detect low numbers of miRNAs in clinical samples is highly valuable but remains a challenge. In this work, we present a novel miRNA detection system by using the elaborately designed hairpin switch, where the T7 primer, template, target recognize sequence, and light-up RNA aptamer template are edited and embedded in one single-stranded DNA hairpin structure. In the beginning, the hairpin switch maintained the hairpin structure 1, in which the ds promoter of T7 polymerase was disrupted, thus the transcription reaction of T7 polymerase was inhibited. After binding to the target, the hairpin switch 1 was unfolded and turned to the hairpin structure 2. This switch initiates the in vitro T7 transcription reaction, producing plenty of RNA transcripts containing RNA aptamers. Consequently, transcribed tremendous RNA aptamers lighted up the fluorophore for quantitative analysis. Compared with the existing T7 polymerase-based amplification system, this strategy exhibits several advantages, including simplicity, convenience, and high selectivity and sensitivity. The experimental results demonstrated that we could achieve the quantification of miRNA in buffer and complex biological samples.

“…Apart from causing cis-cleavage of target DNA, Cas12a undergoes conformational changes upon activation, leading to trans-cleavage activity, which involves the nonspecific cleavage of arbitrary single-stranded DNA . The nucleic acid biosensor built with this feature can not only achieve specific recognition but also swift amplification of nucleic acid signals through efficient cutting. − …”

Section: Introductionmentioning

confidence: 99%

Low-Background Signal-On Homogeneous Electrochemiluminescence Biosensor for Hepatitis B Virus Detection Based on the Regulation of the Length of DNA Modified on the Nanoparticles by CRISPR/Cas12a and Hybridization Chain Reaction

Luo,

Huang,

Luo

et al. 2023

In this work, combined with the high amplification efficiency of hybridization chain reaction (HCR), high specificity of the CRISPR/Cas12a system, and convenience of the homogeneous electrochemiluminescence (ECL) assay based on the regulation of negative charge on the reporting probes, a sensitive ECL biosensor for hepatitis B virus DNA (chosen as a model target) had been developed. The initiator chain trigger DNA that can induce HCR amplification is modified on the surface of ruthenium bipyridine-doped silica nanoparticles (Ru@SiO 2 NPs) first, and large amounts of negative charges modified on the particles were achieved through the HCR amplification reaction. The efficiency of the nanoparticles reaching the negatively charged working electrode can be regulated and realize the change of the ECL signal. In addition, long DNA on the surface of the luminescent body may prevent the coreactant from entering the pore to react with ruthenium bipyridine. These factors combine to produce a low-background system. The presence of the target can activate the CRISPR/Cas12a system and make trigger DNA disappear from the nanoparticle surface, and strong ECL can be detected. The sensor does not require a complex electrode modification; therefore, it has better reproducibility. Additionally, due to dual signal amplification, the sensor has a high sensitivity. In the range of 10 fM to 10 nM, the ECL intensity exhibits a strong linear relationship with the logarithm of the target concentration, and the detection limit is 7.41 fM. This sensor has shown high accuracy in detecting clinical samples, which holds significant potential for application in clinical testing.