CRISPR-Cas12a coupled with terminal deoxynucleotidyl transferase mediated isothermal amplification for sensitive detection of polynucleotide kinase activity - Details

author：

Indexed by：

Abstract：

Polynucleotide　kinase　(PNK)　is　an　important　DNA　damage　repair-related　enzyme　and　also　a　promising　therapeutic　target　in　various　diseases.　Here,　we　developed　a　dual　amplified　sensing　strategy　based　on　the　combination　of　terminal　deoxynucleotidyl　transferase　(TdT)　and　CRISPR-Cas12a　for　high　selective　and　sensitive　detection　of　PNK　activity　in　cell　lysates　and　also　inhibitor　screening.　In　this　sensing　system,　the　PNK　converted　the　3′-phosphate　DNA　to　3′-hydroxy　DNA,　and　then　the　TdT　catalyzed　to　add　a　long　poly-adenine　(poly　(A))　at　the　3′−OH　terminus　DNA　(as　first　amplified　step).　The　produced　poly　(A)　tail　acted　as　activator　to　trigger　the　Cas12a　trans-cleavage　(non-target)　activity　to　cleave　reporter　probes　for　fluorescence　detection　(as　second　amplified　step).　The　dual　amplified　steps　lead　to　a　synergetic　signal　amplification　effect　for　sensitive　detection　of　PNK　activity.　The　developed　sensing　platform　enable　detect　PNK　activity　with　linear　range　from　0　to　1　U/mL　and　a　direct　detection　limit　of　5　×　10−4　U/mL.　It　was　also　successfully　used　for　PNK　inhibitor　screening　and　PNK　activity　detection　in　cell　extracts　even　at　a　single-cell　level.　The　efficient　dual　amplified　method　can　broaden　the　CRISPR/Cas-based　sensing　systems　in　other　bio-/chem-analysis　fields.　©　2020　Elsevier　B.V.

Keyword：

Chemical detection Diagnosis DNA Enzymes Nucleotides

Community：

[ 1 ] [Zhang, Xiaolong]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou; 350025, China
[ 2 ] [Zhang, Xiaolong]Mengchao Med-X Center, Fuzhou University, Fuzhou; 350116, China
[ 3 ] [Zheng, Cheng]College of Chemistry, Fuzhou University, Fuzhou; 350116, China
[ 4 ] [Ding, Lei]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou; 350025, China
[ 5 ] [Ding, Lei]Mengchao Med-X Center, Fuzhou University, Fuzhou; 350116, China
[ 6 ] [Ding, Lei]College of Biological Science and Engineering, Fuzhou University, Fuzhou; 350116, China
[ 7 ] [Wu, Yanni]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou; 350025, China
[ 8 ] [Wu, Yanni]Mengchao Med-X Center, Fuzhou University, Fuzhou; 350116, China
[ 9 ] [Wu, Yanni]College of Biological Science and Engineering, Fuzhou University, Fuzhou; 350116, China
[ 10 ] [Xu, Haipo]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou; 350025, China
[ 11 ] [Xu, Haipo]Mengchao Med-X Center, Fuzhou University, Fuzhou; 350116, China
[ 12 ] [Sun, Yupeng]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou; 350025, China
[ 13 ] [Sun, Yupeng]Mengchao Med-X Center, Fuzhou University, Fuzhou; 350116, China
[ 14 ] [Zeng, Yongyi]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou; 350025, China
[ 15 ] [Zeng, Yongyi]Mengchao Med-X Center, Fuzhou University, Fuzhou; 350116, China
[ 16 ] [Zeng, Yongyi]Liver Disease Center, The First Affiliated Hospital of Fujian Medical University, Fuzhou; 350005, China
[ 17 ] [Liu, Xiaolong]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou; 350025, China
[ 18 ] [Liu, Xiaolong]Mengchao Med-X Center, Fuzhou University, Fuzhou; 350116, China
[ 19 ] [Liu, Xiaolong]Liver Disease Center, The First Affiliated Hospital of Fujian Medical University, Fuzhou; 350005, China
[ 20 ] [Liu, Jingfeng]The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou; 350025, China
[ 21 ] [Liu, Jingfeng]Mengchao Med-X Center, Fuzhou University, Fuzhou; 350116, China
[ 22 ] [Liu, Jingfeng]Liver Disease Center, The First Affiliated Hospital of Fujian Medical University, Fuzhou; 350005, China
[ 23 ] [Liu, Jingfeng]Fujian Medical University Cancer Hospital, Fuzhou; 350014, China

Reprint 's Address：

Email：

Show more details

Related Keywords：

Multilegged Exosomal Walker for Electrochemical Detection of Exosomes
2019，Analytical Chemistry
Metal–Organic Frameworks in Surface Enhanced Raman Spectroscopy–Based Analysis of Volatile Organic Compounds
2024，Advanced Science
Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design
2024，ACS Applied Materials and Interfaces
Au(III)-promoted magnetic molecularly imprinted polymer nanospheres for electrochemical determination of streptomycin residues in food
2013，Biosensors and Bioelectronics
Fiber Optic Sensor Coated with Multiple Layers of Hexagonal Boron Nitride Nanosheets (BNNS) for the Detection of Volatile Organic Compounds
2024，ACS Applied Materials and Interfaces

Source ：

Sensors and Actuators, B: Chemical

ISSN： 0925-4005

Year： 2021

Volume： 330

6 . 3 9 3

JCR@2018

CAS Journal Grade：1

Cited Count：

WoS CC Cited Count：

SCOPUS Cited Count： 26

ESI Highly Cited Papers on the List： 0 Unfold All

WanFang Cited Count：

Chinese Cited Count：

30 Days PV： 0

Affiliated Colleges：

Get Fulltext

DOI Library Discovery Baidu Scholar Search Engineering Village

Type
Departments

All Years Choose Year From to