An all-in-one enzymatic DNA network based on catalytic hairpin assembly for label-free and highly sensitive detection of APE1.

Apurinic/apyrimidinic endonuclease 1 (APE1), identified as a prospective cancer biomarker, plays a vital role in the occurrence and progression of cancer cell lines and impacts on genome stability. However, conventional approaches typically rely on the interactions between the antigen and antibody, limiting their utility for qualitative assessments of APE1 expression. Herein, an all-in-one enzymatic DNA network (EDN) assay with catalytic hairpin assembly for label-free and ultrasensitive detection of APE1 has been developed. In this work, the blocking strand can inhibit the initiator by obstructing the complementary region, preventing the hairpin from hybridizing in the absence of APE1 targets. While the presence of targets can activate the unlocking of the initiator, which can trigger the catalytic hairpin reaction, and increase the fluorescent signal. Under optimal conditions, the developed sensing method can detect the target APE1 down to 4.78 × 10-6 U mL-1 with a wide linear range from 5 × 10-6 U mL-1 to 30 U mL-1. This strategy has also been successfully applied to the analysis of complicated biological samples compared to ELISA, demonstrating its potential applications in biochemical and molecular biology research as well as clinical diagnostics. Overall, benefiting from the high amplification efficiency, this strategy has successfully and simply detected low-abundance APE1 without additional enzyme isolation steps, presenting great potential for clinical detection applications.

An ultrasensitive biosensing platform for FEN1 activity detection based on target-induced primer extension to trigger the collateral cleavage of CRISPR/Cas12a.

Flap endonuclease 1 (FEN1), a structure-selective endonuclease essential for DNA replication and repair, has been considered as a new promising marker for early cancer diagnosis. However, reliable, sensitive and convenient biosensors for FEN1 detection are still technically challenging. Herein, a fluorometric biosensor based on target-induced primer extension to initiate the collateral cleavage of CRISPR/Cas12a has been established for ultrasensitive and specific detection of FEN1 activity. Using branched DNA to probe FEN1 activity, the cleaved 5' flap initiated DNA polymerase-mediated primer extension to produce plenty of DNA duplexes containing protospacer adjacent motif (PAM) which act as activators to initiate the collateral cleavage activity of Cas12a protein, producing an significantly amplified fluorescence response for ultrasensitive determination of FEN1 activity. The developed biosensing platform displays excellent analytical performance, with a limit of detection (LOD) down to 8.9 × 10-5 U µL-1, and a wide linear range from 1.0 × 10-4 to 5.0 × 10-1 U µL-1. Moreover, the proposed strategy was successfully used for FEN1 detection in serums and cell lysates and suggests potential clinical applications, which may provide a reliable approach for FEN1 that will allow effective diagnosis in the early stages of related cancer.

Integrating CRISPR-Cas12a with a crRNA-Mediated Catalytic Network for the Development of a Modular and Sensitive Aptasensor.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, which exhibits excellent target DNA-activated trans-cleavage activity under the guidance of a programmable CRISPR RNA (crRNA), has shown great promise in next-generation biosensing technology. However, current CRISPR-Cas12a-based biosensors usually improve sensitivity by the initial nucleic acid amplification, while the distinct programmability and predictability of the crRNA-guided target binding process has not been fully exploited. Herein, we, for the first time, propose a modular and sensitive CRISPR-Cas12a fluorometric aptasensor by integrating an enzyme-free and robust crRNA-mediated catalytic nucleic acid network, namely, Cas12a-CMCAN, in which crRNA acts as an initiator to actuate cascade toehold-mediated strand displacement reactions (TM-SDRs). As a proof of concept, adenosine triphosphate (ATP) was selected as a model target. Owing to the multiturnover of CRISPR-Cas12a trans-cleavage and the inherent recycling amplification network, this method achieved a limit of detection value of 0.16 µM (20-fold lower than direct Cas12a-based ATP detection) with a linear range from 0.30 to 175 µM. In addition, Cas12a-CMCAN can be successfully employed to detect ATP levels in diluted human serum samples. Considering the simplicity, sensitivity, and easy to tune many targets by changing aptamer sequences, the Cas12a-CMCAN sensing method is expected to offer a heuristic idea for the development of CRISPR-Cas12a-based biosensors and unlock its potential for general and convenient molecule diagnostics.

Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following ß­catenin silencing.

Resistance to the chemotherapeutic drug cisplatin has been documented in various types of cancer, while the increased expression of ß­catenin has been observed in cisplatin­resistant ovarian cancer. However, the involvement of ß­catenin in cisplatin resistance is unclear. The present study investigated the antitumor effect of cisplatin on the proliferation, invasion and apoptosis of breast cancer (BC) cells following ß­catenin silencing in BC, which is the most frequent type of malignancy among women. The expression of ß­catenin in BC tissues and cell lines was measured by reverse transcription­quantitative polymerase chain reaction, and the association between expression levels and clinical characteristics was statistically analyzed. The viability of BC cell lines treated with siR­ß­catenin or with siR­ß­catenin and cisplatin in combination was determined using a Cell Counting Kit­8 assay. The migratory and invasive abilities of BC cells treated with both siR­ß­catenin and cisplatin were examined with Transwell assays. The CD44 antigen/intercellular adhesion molecule 1 expression ratio, cell cycle distribution and apoptosis levels of BC cells treated with siR­ß­catenin and cisplatin in combination were detected by flow cytometry. The expression levels of apoptosis­associated proteins, including caspase­3/9, in the BC cells treated with both siR­ß­catenin and cisplatin were investigated by western blot analysis. The levels of apoptosis in the BC cells following combined treatment with siR­ß­catenin and cisplatin was further quantified by Hoechst 33342 staining. ß­catenin was identified to be highly expressed in BC tissues and cell lines and was associated with pathological stage and lymph node status. Following knockdown of ß­catenin expression, cisplatin treatment suppressed the viabilities, and the migratory and invasive capabilities of the T47D and MCF­7 cells, and induced extensive apoptosis. ß­catenin knockdown upregulated caspase­3/9 levels following cisplatin treatment and induced the apoptosis of T47D and MCF­7 cells. In conclusion, ß­catenin may be of value as a therapeutic target during cisplatin treatment in patients with BC treated with cisplatin.