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Start Over Author "Fan, Gao" Journal analytical chemistry
23 results on '"Fan, Gao"'

6. Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a

Author

Li, Qian, Song, Zhi-Ling, Zhang, Yuxi, Zhu, Lina, Yang, Qian, Liu, Xingfu, Sun, Xufeng, Chen, Xuxu, Kong, Rongmei, Fan, Gao-Chao, and Luo, Xiliang

Abstract

CRISPR/Cas12a has been believed to be powerful in molecular detection and diagnostics due to its amplified trans-cleavage feature. However, the activating specificity and multiple activation mechanisms of the Cas12a system are yet to be elucidated fully. Herein, a “synergistic activator effect” is discovered, which supports an activation mechanism that a synergistic incorporation of two short ssDNA activators can promote the trans-cleavage of CRISPR/Cas12a, while either of them is too short to work independently. As a proof-of-concept example, the synergistic activator-triggered CRISPR/Cas12a system has been successfully harnessed in the AND logic operation and the discrimination of single-nucleotide variants, requiring no signal conversion elements or other amplified enzymes. Moreover, a single-nucleotide specificity has been achieved for the detection of single-nucleotide variants by pre-introducing a synthetic mismatch between crRNA and the “helper” activator. The finding of “synergistic activator effect” not only provides deeper insight into CRISPR/Cas12a but also may facilitate its expanded application and power the exploration of the undiscovered properties of other CRISPR/Cas systems.

Published
2023
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10. DNA-Templated Aptamer Probe for Identification of Target Proteins

Author

Enguo Fan, Guijin Zhai, Ye Wang, Xue Bai, Shanshan Tian, Congcong Lu, Kai Zhang, Fan Gao, Yukui Zhang, and Wenjing Bi

Subjects
0301 basic medicine, Chemistry, Binding protein, Aptamer, DNA, Single-Stranded, Aptamers, Nucleotide, 010402 general chemistry, 01 natural sciences, Molecular biology, Protein markers, 0104 chemical sciences, Analytical Chemistry, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, Egg White, Biochemistry, Covalent bond, Complementary DNA, Muramidase, Biomarker discovery, DNA Probes, Molecular probe, DNA
Abstract

Using aptamers as molecular probes for biomarker discovery has attracted a great deal of attention in recent years. However, it is still a big challenge to accurately identify those protein markers that are targeted by aptamers under physiological conditions due to weak and noncovalent aptamer-protein interactions. Herein, we developed an aptamer based dual-probe using DNA-templated chemistry and photo-cross-linking technique for the identification of target proteins that are recognized by aptamers. In this system, the aptamer was modified by a single strand DNA as binding probe (BP), and another complementary DNA with a photoactive group and reporter group was modified as capture probe (CP). BP was first added to recruit the binding protein via aptamer recognition, and subsequently CP was added to let the cross-linker close to the target via DNA self-assembly, and then a covalent bond between CP and its binding protein was achieved via photo-cross-linking reaction. The captured protein can be detected or affinity enrichment using the tag, finally identified by MS. By use of lysozyme as a model substrate, we demonstrated that this multiple functionalized probe can be utilized for a successful labeling and enrichment of target protein even under a complicated and real environment. Thus, a novel method to precisely identify the aptamer-targeted proteins has been developed and it has a potential application for discovery of aptamer-based biomarkers.

Published
2017

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