Original Articles

Enzyme-free, Colorimetric DNA Detection System Powered by Autocatalytic Assembly Circuits and G-quadruplex DNAzymes

Abstract

Objectives: To develop an enzyme-free colorimetric DNA detection system combining autocatalytic assembly circuits with G-quadruplex DNAzymes for highly sensitive and visual DNA identification.

Methods: The platform utilized toehold-mediated strand displacement (TMSD) integrated with autocatalytic hairpin circuits and a G-quadruplex DNAzyme. Buffer conditions, hemin concentration, temperature, and reaction time were optimized. Spectrophotometric detection at 416 nm was employed, and NUPACK software was used for oligonucleotide design validation.

Results: The optimized system exhibited a detection limit as low as 31 pM. Clear electrophoretic band patterns confirmed correct structure formation. The system showed high reproducibility, sensitivity, and a visible signal without enzymatic components.

Conclusions: This detection method is simple, cost-effective, and highly sensitive, with potential for point-of-care diagnostics, food safety testing, and environmental monitoring. The enzyme-free nature, label-free operation, and modular design make it adaptable to other nucleic acid targets.   

1. Walbrun A, Wang T, Matthies M, Šulc P, Simmel FC, Rief M. Single-molecule force spectroscopy of toehold-mediated strand displacement. Nat Commun. 2024;15(1):7564. https://doi.org/10.1038/s41467-024-51813-9
2. Yue S, Song X, Song W, Bi S. An enzyme-free molecular catalytic device: dynamically self-assembled DNA dendrimers for in situ imaging of microRNAs in live cells. Chem Sci. 2019;10(6):1651-8. https://doi.org/10.1039/c8sc04756a
3. Tang L, Luo T, Fan S, Liu Y, Song J. Principles of nucleic acid toehold mediated strand displacement (TMSD) reaction model and its applications in cell environment. Biomater Sci. 2023;11(15):5060-77. https://doi.org/10.1039/d3bm00476g
4. Tan K, Chen L, Cao D, Xiao W, Lv Q, Zou L. A rapid and highly sensitive ctDNA detection platform based on locked nucleic acid-assisted catalytic hairpin assembly circuits. Anal Methods. 2023;15(35):4548-54. https://doi.org/10.1039/d3ay01150j
5. Deng W, Xu JY, Peng H, Huang CZ, Le XC, Zhang H. DNAzyme motor systems and logic gates facilitated by toehold exchange translators. Biosens Bioelectron. 2022;217:114704. https://doi.org/10.1016/j.bios.2022.114704
6. Liu Y, Jin L, Mao J, Deng R, Lin F, Cheng Y, et al. Signal amplified colorimetric nucleic acid detection based on autocatalytic hairpin assembly. RSC adv. 2024;14(24):17152-7. https://doi.org/10.1039/d4ra01982b
7. Fornace ME, Huang J, Newman CT, Porubsky NJ, Pierce MB, Pierce NA. NUPACK: analysis and design of nucleic acid structures, devices, and systems. 2022. https://doi.org/10.26434/chemrxiv-2022-xv98l
8. Jiang Y, Li B, Milligan JN, Bhadra S, Ellington AD. Real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly. J Am Chem Soc. 2013;135(20):7430-3. https://doi.org/10.1021/ja4023978
9. Wang X-P, Yin B-C, Wang P, Ye B-C. Highly sensitive detection of microRNAs based on isothermal exponential amplification-assisted generation of catalytic G-quadruplexDNAzyme. Biosens Bioelectron. 2013;42:131-5. https://doi.org/10.1016/j.bios.2012.10.097
10. Liu J, Zhang Y, Xie H, Zhao L, Zheng L, Ye H. Applications of catalytic hairpin assembly reaction in biosensing. Small. 2019;15(42):1902989. https://doi.org/10.1002/smll.201902989
11. Karunanayake Mudiyanselage AP, Yu Q, Leon-Duque MA, Zhao B, Wu R, You M. Genetically encoded catalytic hairpin assembly for sensitive RNA imaging in live cells. J Am Chem Soc. 2018;140(28):8739-45. https://doi.org/10.1021/jacs.8b03956
12. Li R, Zhu Y, Gong X, Zhang Y, Hong C, Wan Y, et al. Self-stacking autocatalytic molecular circuit with minimal catalytic DNA assembly. J Am Chem Soc. 2023;145(5):2999-3007. https://doi.org/10.1021/jacs.2c11504
13. Yang Y, Chen R, Guo Y, Zhang J, Ren S, Zhou H, et al. A two-color fluorescence sensing strategy based on functionalized tetrahedral DNAzyme nanotweezers for ochratoxin A detection. Talanta. 2025;285:127348. https://doi.org/10.1016/j.talanta.2024.127348
Files
IssueVol 3 No 1 (2025) QRcode
SectionOriginal Articles
DOI https://doi.org/10.18502/abi.v3i1.19349
Keywords
DNA Nanotechnology DNAzyme TMSD Colorimetric Technique
Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
1.
Davood R, Ravan H, Shahram S. Enzyme-free, Colorimetric DNA Detection System Powered by Autocatalytic Assembly Circuits and G-quadruplex DNAzymes. ABI. 2025;3(1):36-45.