4.7 Article

Target-mediated assembly formation of multi-arm DNAzyme nanostructures for sensitive and accurate discrimination of single-nucleotide polymorphism in K-ras gene

Journal

SENSORS AND ACTUATORS B-CHEMICAL
Volume 346, Issue -, Pages -

Publisher

ELSEVIER SCIENCE SA
DOI: 10.1016/j.snb.2021.130535

Keywords

Single-nucleotide polymorphism; Fluorescent detection; DNAzyme; G-quadruplex; Signal amplification

Funding

  1. National Natural Science Foundation of China [22004010]
  2. Science and Technology Research Program of Chongqing Municipal Education Commission [KJQN201901135]
  3. Chongqing Science and Technology Commission of China [cstc2019jcyj-msxmX0196]
  4. Scientific Research Foundation of Chongqing University of Technology

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A non-enzymatic and label-free sensing method is developed for accurate and ultrasensitive differentiation of SNP in the K-ras gene, showing high sensitivity with a detection limit of 3.63 fM and high selectivity to discriminate MtDNA from a mixture with a concentration ratio of WtDNA to MtDNA as high as 5000:1. This approach has the potential application for future clinical diagnostics by analyzing target MtDNA in diluted serum samples.
Single-nucleotide polymorphism (SNP) is a typical way of genetic variation and commonly used as genetic marker to indicate various types of human diseases. We herein report a non-enzymatic and label-free sensing method for accurate and ultrasensitive differentiation of SNP in K-ras gene. The presence of target mutant K-ras gene (MtDNA) leads to the formation of three-way junctions (3WJ) structure with an exposure toehold site, which initiates the toehold strand displacement reaction (TSDR) to form multi-arm DNAzyme nanostructures. These DNAzymes further cyclically cleave the hairpin DNA substrates and release many G-rich fragments to interact with organic dye thioflavin T (ThT) and yield significantly enhanced fluorescence for sensitive discrimination of MtDNA. However, the presence of wild K-ras gene (WtDNA) trigger a base pairing frame shift on the 3WJ structure and result in the closure of the toehold site, thus neither TSDR nor DNAzyme-assistant cleavage reaction is initiated, and no obvious fluorescent signal is observed. Importantly, this strategy shows high sensitivity with a detection limit of 3.63 fM and exhibits high selectivity to discriminate MtDNA from a mixture with a concentration ratio of WtDNA to MtDNA as high as 5000:1. Moreover, this approach can be used to analyze target MtDNA in diluted serum samples, which demonstrates its potential application for future clinical diagnostics.

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