Multimodal detection of flap endonuclease 1 activity through CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides.

Biosens Bioelectron

Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong Special Administrative Region, China; City University of Hong Kong Shenzhen Research Institute, Shenzhen, China. Electronic address:

Published: January 2023

Flap endonuclease 1 (FEN1) is an endonuclease that specially removes 5' single-stranded overhang of branched duplex DNA (5' flap). While FEN1 is essential in various DNA metabolism pathways for preventing the malignant transformation of cells, an unusual expression of FEN1 is often associated with tumor progression, making it a potential biomarker for cancer diagnosis and treatment. Here we report a multimodal detection of FEN1 activity based on CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides (ssDNA). A dumbbell DNA structure with a 5' flap was designed, which can be cleaved by the FEN1 and the dumbbell DNA is subsequently ligated by T4 DNA ligase. The resulting closed duplex DNA contains a specific protospacer adjacent motif (PAM) that activates trans-cleavage of ssDNA after binding to CRISPR/Cas12a-crRNA. The trans-cleavage is activated only once and is independent to length or sequence of the ssDNA, which allows efficient signal amplification and multimodal signals such as fluorescence or cleaved connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that alters solution turbidity after magnetic separation. In addition, by loading the particle solution into a microfluidic chip, unconnected PMPs escaping from a magnetic separator are amassed at the particle dam, enabling a visible PMP accumulation length proportional to the FEN1 activity. This multimodal detection is selective to FEN1 and achieves a low limit of detection (LOD) with only 40 min of reaction time. Applying to cell lysates, higher FEN1 activity was detected in breast cancer cells, suggesting a great potential for cancer diagnosis.

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bios.2022.114859DOI Listing

Publication Analysis

Top Keywords

multimodal detection
12
fen1 activity
12
flap endonuclease
8
crispr/cas12a trans-cleavage
8
trans-cleavage single-strand
8
dna
8
single-strand dna
8
dna oligonucleotides
8
fen1
8
duplex dna
8

Similar Publications

Background: To reduce the mortality related to bladder cancer, efforts need to be concentrated on early detection of the disease for more effective therapeutic intervention. Strong risk factors (eg, smoking status, age, professional exposure) have been identified, and some diagnostic tools (eg, by way of cystoscopy) have been proposed. However, to date, no fully satisfactory (noninvasive, inexpensive, high-performance) solution for widespread deployment has been proposed.

View Article and Find Full Text PDF

This review assesses the primary neuroimaging techniques used to evaluate Parkinson's disease (PD) - a neurological condition characterized by gradual dopamine-producing nerve cell degeneration. The neuroimaging techniques explored include positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). These modalities offer varying degrees of insights into PD pathophysiology, diagnostic accuracy, specificity by way of exclusion of other Parkinsonian syndromes, and monitoring of disease progression.

View Article and Find Full Text PDF

Background: Traditional 2-dimensional (2D) ultrasound is a noninvasive method in the assessment of glomerular disease. Ultrasound elastography shows promise in evaluating renal fibrosis, which plays a key role in glomerular disease progression. However, research in pediatric cohorts is limited.

View Article and Find Full Text PDF

Background: The ability of conventional ultrasound (US)-guided liver biopsy to visualize certain liver lesions, particularly those affected by conditions like hepatitis or cirrhosis, which can obscure lesion boundaries and lead to inaccurate biopsy targeting, is limited. This study aimed to evaluate the potential of multimodal US techniques to improve the visibility of liver lesions that are indistinct under conventional US, and to enhance the success rate of percutaneous biopsies.

Methods: In total, 144 patients with liver masses and lesions that were not clearly visible on conventional US from October 2018 to January 2024 were enrolled in this retrospective analysis.

View Article and Find Full Text PDF

Background: Extrahepatic Common Bile Duct Obstruction (EHBDO) is a serious condition that requires accurate diagnosis for effective treatment. Magnetic Resonance Cholangiopancreatography (MRCP) is a widely used noninvasive imaging technique for visualizing bile ducts, but its interpretation can be complex.

Objective: This study aimed to develop a deep learning-based classification model that integrates MRCP images and clinical parameters to assist radiologists in diagnosing EHBDO more accurately.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!