Exploring the Trans-Cleavage Activity with Rolling Circle Amplification for Fast Detection of miRNA.

MicroRNAs (miRNAs) are a class of endogenous short noncoding RNA. They regulate gene expression and function, essential to biological processes. It is necessary to develop an efficient detection method to determine these valuable biomarkers for the diagnosis of cancers. In this paper, we proposed a general and rapid method for sensitive and quantitative detection of miRNA by combining CRISPR-Cas12a and rolling circle amplification (RCA) with the precircularized probe. Eventually, the detection of miRNA-21 could be completed in 70 min with a limit of detection of 8.1 pM with high specificity. The reaction time was reduced by almost 4 h from more than 5 h to 70 min, which makes detection more efficient. This design improves the efficiency of CRISPR-Cas and RCA-based sensing strategy and shows great potential in lab-based detection and point-of-care test.

CRISPR-Cas12a-assisted elimination of the non-specific signal from non-specific amplification in the Exponential Amplification Reaction.

Non-specific amplification is a major problem in nucleic acid amplification resulting in false-positive results, especially for exponential amplification reactions (EXPAR). Although efforts were made to suppress the influence of non-specific amplification, such as chemical blocking of the template's 3'-ends and sequence-independent weakening of template-template interactions, it is still a common problem in many conventional EXPAR reactions. In this study, we propose a novel strategy to eliminate the non-specific signal from non-specific amplification by integrating the CRISPR-Cas12a system into two-templates EXPAR. An EXPAR-Cas12a strategy named EXPCas was developed, where the Cas12a system acted as a filter to filter out non-specific amplificons in EXPAR, suppressing and eliminating the influence of non-specific amplification. As a result, the signal-to-background ratio was improved from 1.3 to 15.4 using this method. With microRNA-21 (miRNA-21) as a target, the detection can be finished in 40 min with a LOD of 103 fM and no non-specific amplification was observed.

Capture-SELEX of DNA Aptamers for Estradiol Specifically and Estrogenic Compounds Collectively.

Estrogenic compounds such as estrone (E1), 17ß-estradiol (E2), and 17α-ethynylestradiol (EE2) are serious environmental contaminants due to their potent biological activities. At least six selections were previously reported to obtain DNA aptamers for E2, highlighting its environmental importance. A careful analysis revealed that the previous aptamers either are too long or do not bind optimally. Herein, a series of new aptamers were obtained from the capture-SELEX method with dissociation constants down to 30 nM as determined by isothermal titration calorimetry (ITC). Two aptamers were converted to structure-switching fluorescent biosensors, which achieved a limit of detection down to 3.3 and 9.1 nM E2, respectively. One aptamer showed similar binding affinities to all the three estrogens, while the other aptamer is more selective for E2. Both aptamers required Mg2+ for binding. The proposed sensors were successfully applied in the determination of E2 in wastewater. Moreover, comparisons were made with previous aptamers based on primary sequence alignment and secondary structures. Among previously reported truncated aptamers, ITC showed binding only in one of them. The newly selected aptamers have the combined advantages of small size and high affinities.

Aptamer assisted CRISPR-Cas12a strategy for small molecule diagnostics.

Molecular diagnostics are vital for the identification, prevention, and treatment of numerous diseases and are of particular demand in point-of-care (POC) settings. Nevertheless, most reported biosensors based on the CRISPR-Cas system have focused on nucleic-acid targets. Here, we report a versatile diagnostic strategy for small molecules called Molecular Radar (Random Molecular Aptamer-Dependent CRISPR-Assist Reporter), The workflow is simple, convenient, and rapid (conducted at 37 °C in under 25 min), indicating the substantial potential of the proposed assay could be adapted into a biosensor for POC settings and on-site molecular diagnostics. This strategy is based on the CRISPR Cas12a-assisted fluorescence reporter system that consists of Cas12a, CRISPR RNA (crRNA), a single-stranded DNA (ssDNA) probe labeled with a fluorophore at the 5' end and a quencher at the 3' end (F-Q probe), and a single-stranded DNA aptamer for the target molecule. In the presence of a target molecule, the aptamer binds to this small molecule with high specificity and affinity, resulting in a decrease of aptamer hybridized to the crRNA-Cas12a duplex. This decrease in activated Cas12a leads to a significant reduction in fluorescence signal. In this study, adenosine-5'-triphosphate (ATP) was selected as model target molecule and an ATP detect method was developed with high specificity and sensitivity with a linear range from 25 to 500 µM and a detection limit of 104 nM. Moreover, the particular characteristics of CRISPR-Cas12a that we report here for the first time have enriched our understanding of Cas12a and provided guidance for further research on CRISPR-Cas12a-based biosensors.

A 'turn-on' ultra-sensitive multiplex real-time fluorescent quantitative biosensor mediated by a universal primer and probe for the detection of genetically modified organisms.

Among the existing multiplex genetically modified organism (GMO) detection methods, significant problems are highlighted, including amplification asymmetry of different targets, and the low detection throughput, which limits their capacity to meet the requirements of high-throughput analysis. To mitigate these challenges, a 'turn-on' ultra-sensitive multiplex real-time fluorescent quantitative biosensor is developed. In this system, the multiplex ligation-dependent amplification (MLPA), universal primer and universal probe are innovatively combined, which can enhanced the amplification specificity, overcome asymmetric amplification and guarantee the homogeneity of amplification efficiency simultaneously. Furthermore, both single and multiplex detection results can be output by the fluorescent group labeled on universal TaqMan probes for different targets in real-time. After optimization, the quantitative detection limit was 5 pg. In conclusion, this strategy could serve as an important tool for GMO detection in processed and commercially available products, even in the fields that require reliable and sensitive detection of DNA targets.

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.