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Structure-switchable branched inhibitors regulate the activity of CRISPR-Cas12a for nucleic acid diagnostics.
Anal Chim Acta
January 2025
Department of Laboratory Medicine, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, People's Republic of China; Wuhan Research Center for Infectious Diseases and Cancer, Chinese Academy of Medical Sciences, Wuhan, People's Republic of China; Hubei Engineering Center for Infectious Disease Prevention, Control and Treatment, Wuhan, People's Republic of China. Electronic address:
Background: In current years, the CRISPR (clustered regularly interspaced short palindromic repeats) based strategies have emerged as the most promising molecular tool in the field of gene editing, intracellular imaging, transcriptional regulation and biosensing. However, the recent CRISPR-based diagnostic technologies still require the incorporation of other amplification strategies (such as polymerase chain reaction) to improve the cis/trans cleavage activity of Cas12a, which complicates the detection workflow and lack of a uniform compatible system to respond to the target in one pot.
Results: To better fully-functioning CRISPR/Cas12a, we reported a novel technique for straightforward nucleic acid detection by incorporating enzyme-responsive steric hindrance-based branched inhibitors with CRISPR/AsCas12a methodology.
DNAzyme assisted single amplification for FEN1 activity detection using a personal glucose meter.
Anal Chim Acta
January 2025
MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, 350116, PR China. Electronic address:
Flap endonuclease 1 (FEN1) plays a vital role in cancer by modulating DNA repair mechanisms, inducing genomic instability, and serving as a promising biomarker for cancer diagnosis and prognosis. In this work, we present the development of a novel DNAzyme signal amplification-directed point-of-care sensing system (Dz-PGM) for the sensitive and specific detection of FEN1. The Dz-PGM system utilizes DNAzyme signal amplification in conjunction with a personal glucose meter (PGM) for reporting, capitalizing on a biochemical cascade initiated by FEN1 recognition.
View Article and Find Full Text PDFRational Design of a Novel DNA Polymerase From Clostridium thermocellum to Improve LAMP Detection Performance.
Biotechnol J
January 2025
School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Loop-mediated isothermal amplification (LAMP) is a detection method widely used in pathogen detection and clinical diagnosis. Nevertheless, it is highly constrained by thermal stability, catalytic activity, and resistance to inhibitors of Bst DNA polymerase. In this study, a novel DNA polymerase was characterized from Clostridium thermocellum, exhibiting potential in LAMP detection.
View Article and Find Full Text PDFAmplification-free CRISPR/Cas based dual-enzymatic colorimetric nucleic acid biosensing device.
Lab Chip
January 2025
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
Nucleic acid testing (NAT) is widely considered the gold standard in analytical fields, with applications spanning environmental monitoring, forensic science and clinical diagnostics, among others. However, its widespread use is often constrained by complicated assay procedures, the need for specialized equipment, and the complexity of reagent handling. In this study, we demonstrate a fully integrated 3D-printed biosensensing device employing a CRISPR/Cas12a-based dual-enzymatic mechanism for highly sensitive and user-friendly nucleic acid detection.
View Article and Find Full Text PDFSelection of a Fluorinated Aptamer Targeting the Viral RNA Frameshift Element with Different Chiralities.
Biochemistry
January 2025
Department of Biochemistry and Molecular Biology, Melvin and Bren Simon Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States.
The development of RNA aptamers with high specificity and affinity for target molecules is a critical advancement in the field of therapeutic and diagnostic applications. This study presents the selection of a 2'-fluoro-modified mirror-image RNA aptamer through the in vitro SELEX process. Using a random RNA library, we performed iterative rounds of selection and amplification to enrich aptamers that bind specifically to the viral attenuator hairpin RNA containing the opposite chirality, which is an important part of the frameshift element.
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