Label-free detection of microRNA by polymerization and isomerization cyclic amplification coupled with G/Hemin DNAzyme

Xinlan Zhu; Ziyan Zhang; Ruiyang Ma; Huachu Chen; Yi Zheng; Su Zeng; Zheyong Li; Sheng Cai

doi:10.1016/j.jpha.2025.101536

Article Contents

Article Navigation > Journal of Pharmaceutical Analysis > 2025 > Accepted Manu

Xinlan Zhu, Ziyan Zhang, Ruiyang Ma, Huachu Chen, Yi Zheng, Su Zeng, Zheyong Li, Sheng Cai. Label-free detection of microRNA by polymerization and isomerization cyclic amplification coupled with G/Hemin DNAzyme[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2025.101536

Citation:

Xinlan Zhu, Ziyan Zhang, Ruiyang Ma, Huachu Chen, Yi Zheng, Su Zeng, Zheyong Li, Sheng Cai. Label-free detection of microRNA by polymerization and isomerization cyclic amplification coupled with G/Hemin DNAzyme[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2025.101536

Citation:

Xinlan Zhu, Ziyan Zhang, Ruiyang Ma, Huachu Chen, Yi Zheng, Su Zeng, Zheyong Li, Sheng Cai. Label-free detection of microRNA by polymerization and isomerization cyclic amplification coupled with G/Hemin DNAzyme[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2025.101536

PDF( 5387 KB)

Label-free detection of microRNA by polymerization and isomerization cyclic amplification coupled with G/Hemin DNAzyme

doi: 10.1016/j.jpha.2025.101536

Xinlan Zhu^a,b,
Ziyan Zhang^a,
Ruiyang Ma^a,
Huachu Chen^a,
Yi Zheng^a,b,
Su Zeng^{a,b
,
,},
Zheyong Li^{c
,
,},
Sheng Cai^{a,b
,
,}

a. Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China;
b. College of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China;
c. General surgery department, Sir Run Run Shaw hospital, Alaer hospital, Zhejiang university, Alaer, Xinjiang 843300, China

Funds:

We acknowledge financial support from the Key Research and Development Program funded by the Science and Technology Council of the XinJiang Production and Construction Corps (Program No.: 2023AB018-08), and the National Natural Science Foundation of China (Grant No.: 82373828). The authors thank Nan Zhou from the Core Facilities, Zhejiang University School of Medicine for the technical support.

Received Date: Sep. 23, 2025
Accepted Date: Dec. 20, 2025
Rev Recd Date: Dec. 19, 2025
Available Online: Dec. 23, 2025

Abstract

Abstract

A label-free detection technique for microRNA (miRNA) was developed based on target replacement cycling, polymerization and isomerization cyclic amplification (PICA), and G/Hemin DNAzyme signal output. The designed hairpin probe undergoes cyclic alternation of polymerization and isomerization catalyzed by Bst DNA polymerase, enabling target replacement cycling and continuous self-extension of single-stranded DNA (ssDNA) strands. This process generates abundant G-quadruplex sequences that bind to Hemin, achieving label-free detection of miRNA through catalytic reactions. Its key advantages include a specific primer self-extension mechanism that avoids spurious amplification, one step isothermal design simplifies workflow and enhances system robustness, adapting to diverse detection scenarios, and label-free detection strategy that substantially reduces cost. Therefore, it is poised to become a reliable tool for clinical miRNA analysis, offering potential guidance for the early diagnosis, treatment, and prognosis of tumor biomarkers.
- MicroRNA (miRNA),
- Target replacement cycling,
- Polymerization and isomerization cyclic amplification (PICA),
- G/Hemin DNAzyme,
- Clinical sample

FullText(HTML)

References(48)

References

[1]	M. Ha, V.N. Kim, Regulation of microRNA biogenesis - Nature Reviews Molecular Cell Biology, Nat. Rev. Mol. Cell Biol. 15 (2014) 509-524 https://doi.org/10.1038/nrm3838.
[2]	K. Saliminejad, H.R. Khorram Khorshid, S. Soleymani Fard, et al., An overview of microRNAs: Biology, functions, therapeutics, and analysis methods, J. Cell. Physiol. 234 (2019) 5451-5465 https://doi.org/10.1002/jcp.27486.
[3]	M. Zhong, K. Chen, W. Sun, et al., PCDetection: PolyA-CRISPR/Cas12a-based miRNA detection without PAM restriction, Biosens. Bioelectron. 214 (2022) 114497 https://doi.org/10.1016/j.bios.2022.114497.
[4]	M.T. Quang, M.N. Nguyen, The potential of microRNAs in cancer diagnostic and therapeutic strategies: a narrative review, J. Basic Appl. Zool. 85 (2024) 7 https://doi.org/10.1186/s41936-024-00360-2.
[5]	E. Dissanayake, Y. Inoue, MicroRNAs in Allergic Disease, Curr. Allergy Asthma Rep. 16 (2016) 67 https://doi.org/10.1007/s11882-016-0648-z.
[6]	A. Gaur, D.A. Jewell, Y. Liang, et al., Characterization of MicroRNA Expression Levels and Their Biological Correlates in Human Cancer Cell Lines, Cancer Res. 67 (2007) 2456-2468 https://doi.org/10.1158/0008-5472.CAN-06-2698.
[7]	Y. Shao, J. Xu, W. Chen, et al., miR-135b: An emerging player in cardio-cerebrovascular diseases, J. Pharm. Anal. 14 (2024) 100997 https://doi.org/10.1016/j.jpha.2024.100997.
[8]	M. Zhou, J. Wang, Y. Peng, et al., Elemene as a binding stabilizer of microRNA-145-5p suppresses the growth of non-small cell lung cancer, J. Pharm. Anal. 15 (2025) 101118 https://doi.org/10.1016/j.jpha.2024.101118.
[9]	S. Xu, Y. Chang, Z. Wu, et al., One DNA circle capture probe with multiple target recognition domains for simultaneous electrochemical detection of miRNA-21 and miRNA-155, Biosens. Bioelectron. 149 (2020) 111848 https://doi.org/10.1016/j.bios.2019.111848.
[10]	A.J. Schetter, H. Okayama, C.C. Harris, The Role of MicroRNAs in Colorectal Cancer, Cancer J. 18 (2012) 244 https://doi.org/10.1097/PPO.0b013e318258b78f.
[11]	T.X. Lu, M.E. Rothenberg, MicroRNA, J. Allergy Clin. Immunol. 141 (2018) 1202-1207 https://doi.org/10.1016/j.jaci.2017.08.034.
[12]	T. Kim, C.M. Croce, MicroRNA: trends in clinical trials of cancer diagnosis and therapy strategies, Exp. Mol. Med. 55 (2023) 1314-1321 https://doi.org/10.1038/s12276-023-01050-9.
[13]	J. Song, C.Y. Lee, H.G. Park, Ultrasensitive multiplexed miRNA detection based on a self-priming hairpin-triggered isothermal cascade reaction, Chem. Commun. 58 (2022) 2279-2282 https://doi.org/10.1039/D1CC06282D.
[14]	Q. Sun, X. Lei, X. Yang, CircRNAs as upstream regulators of miRNA//HMGA2 axis in human cancer, Pharmacol. Ther. 263 (2024) 108711 https://doi.org/10.1016/j.pharmthera.2024.108711.
[15]	K. Sato, E. Osaka, K. Fujiwara, et al., miRNA-218 targets multiple oncogenes and is a therapeutic target for osteosarcoma, Oncol. Rep. 47 (2022) 92 https://doi.org/10.3892/or.2022.8303.
[16]	B.M. Hussen, H.J. Hidayat, A. Salihi, et al., MicroRNA: A signature for cancer progression, Biomed. Pharmacother. 138 (2021) 111528 https://doi.org/10.1016/j.biopha.2021.111528.
[17]	M. Budakoti, A.S. Panwar, D. Molpa, et al., Micro-RNA: The darkhorse of cancer, Cell. Signal. 83 (2021) 109995 https://doi.org/10.1016/j.cellsig.2021.109995.
[18]	Y. Jia, A. Zang, Y. Shang, et al., MicroRNA-146a rs2910164 polymorphism is associated with susceptibility to non-small cell lung cancer in the Chinese population, Med. Oncol. 31 (2014) 194 https://doi.org/10.1007/s12032-014-0194-2.
[19]	W. Zhu, Q. Ni, Z. Wang, et al., MiR-101-3p targets the PI3K-AKT signaling pathway via Birc5 to inhibit invasion, proliferation, and epithelial-mesenchymal transition in hepatocellular carcinoma, Clin. Exp. Med. 25 (2025) 88 https://doi.org/10.1007/s10238-025-01622-1.
[20]	X. Feng, Z. Wang, R. Fillmore, et al., MiR-200, a new star miRNA in human cancer, Cancer Lett. 344 (2014) 166-173 https://doi.org/10.1016/j.canlet.2013.11.004.
[21]	T. Shi, Q. Hua, Z. Ma, et al., Downregulation of miR-200a-3p induced by hepatitis B Virus X (HBx) Protein promotes cell proliferation and invasion in HBV-infection-associated hepatocarcinoma, Pathol. Res. Pract. 213 (2017) 1464-1469 https://doi.org/10.1016/j.prp.2017.10.020.
[22]	H. Fu, W. Song, X. Chen, et al., MiRNA-200a induce cell apoptosis in renal cell carcinoma by directly targeting SIRT1, Mol. Cell. Biochem. 437 (2018) 143-152 https://doi.org/10.1007/s11010-017-3102-1.
[23]	Y. Teng, X. Su, X. Zhang, et al., miRNA-200a/c as potential biomarker in epithelial ovarian cancer (EOC): evidence based on miRNA meta-signature and clinical investigations, Oncotarget 7 (2016) 81621 https://doi.org/10.18632/oncotarget.13154.
[24]	Y. Zang, Y. Tai, B. Wan, et al., miR-200a-3p promotes the proliferation of human esophageal cancer cells by post-transcriptionally regulating cytoplasmic collapsin response mediator protein-1, Int. J. Mol. Med. 38 (2016) 1558-1564 https://doi.org/10.3892/ijmm.2016.2758.
[25]	A. Pavlic, K. Urh, K. Stajer, et al., Epithelial-Mesenchymal Transition in Colorectal Carcinoma: Comparison Between Primary Tumor, Lymph Node and Liver Metastases, Front. Oncol. 11 (2021) 662806 https://doi.org/10.3389/fonc.2021.662806.
[26]	X. Li, F. Yang, C. Gan, et al., Sustainable and cascaded catalytic hairpin assembly for amplified sensing of microRNA biomarkers in living cells, Biosens. Bioelectron. 197 (2022) 113809 https://doi.org/10.1016/j.bios.2021.113809.
[27]	R. Li, Y. Zhu, X. Gong, et al., Self-Stacking Autocatalytic Molecular Circuit with Minimal Catalytic DNA Assembly, J. Am. Chem. Soc. 145 (2023) 2999-3007 https://doi.org/10.1021/jacs.2c11504.
[28]	Y. Xue, H. Xie, Y. Wang, et al., Novel and sensitive electrochemical/fluorescent dual-mode biosensing platform based on the cascaded cyclic amplification of enzyme-free DDSA and functional nucleic acids, Biosens. Bioelectron. 218 (2022) 114762 https://doi.org/10.1016/j.bios.2022.114762.
[29]	M. Sun, Q. Zhou, J. Peng, et al., Toehold Strand Displacement-Mediated Exponential HCR for Highly Sensitive and Specific Analysis of miRNA in Living Cells, Anal. Chem. 96 (2024) 9078-9087 https://doi.org/10.1021/acs.analchem.4c00594.
[30]	S. Galimberti, S. Balducci, F. Guerrini, et al., Digital Droplet PCR in Hematologic Malignancies: A New Useful Molecular Tool, Diagnostics (Basel). 12 (2022) 1305 https://doi.org/10.3390/diagnostics12061305.
[31]	G. Xie, Z. Zhan, Y. Ye, et al., Hybrid RCA-DLS assay combined with aPCR for sensitive Salmonella enteritidis detection, Anal. Biochem. 646 (2022) 114647 https://doi.org/10.1016/j.ab.2022.114647.
[32]	C.A. Heid, J. Stevens, K.J. Livak, et al., Real time quantitative PCR., Genome Res. 6 (1996) 986-994 https://doi.org/10.1101/gr.6.10.986.
[33]	X. Huang, X. Wang, X. Wang, et al., Template and skeleton-co-confinement pyrolysis to synthesize two-dimensional N-doped carbon nanoflakes for profiling dynamic antibiotic degradation, J. Hazard. Mater. 495 (2025) 139104 https://doi.org/10.1016/j.jhazmat.2025.139104.
[34]	L. Lan, J. Huang, M. Liu, et al., Polymerization and isomerization cyclic amplification for nucleic acid detection with attomolar sensitivity, Chem. Sci. 12 (2021) 4509-4518 https://doi.org/10.1039/D0SC05457G.
[35]	T.G. Lei Liao, Bingying Jiang, Ruo Yuan, Yun Xiang, Target recycling-triggered polymerization/isomerization amplification cascades for sensitive microRNA assay, Sens. Actuators B 379 (2023) 133221 https://doi.org/10.1016/j.snb.2022.133221.
[36]	X. Yang, C. Fang, H. Mei, et al., Characterization of G-Quadruplex/Hemin Peroxidase: Substrate Specificity and Inactivation Kinetics, Chem. Eur. J. 17 (2011) 14475-14484 https://doi.org/10.1002/chem.201101941.
[37]	X. Cheng, X. Liu, T. Bing, et al., General Peroxidase Activity of G-Quadruplex−Hemin Complexes and Its Application in Ligand Screening, Biochemistry 48 (2009) 7817-7823 https://doi.org/10.1021/bi9006786.
[38]	L. Gu, Y. Ding, Y. Zhou, et al., Selective Hemin Binding by a Non-G-quadruplex Aptamer with Higher Affinity and Better Peroxidase-like Activity, Angew. Chem. Int. Ed. 63 (2024) e202314450 https://doi.org/10.1002/anie.202314450.
[39]	P. Travascio, Y. Li, D. Sen, DNA-enhanced peroxidase activity of a DNA aptamer-hemin complex, Chem. Biol. 5 (1998) 505-517 https://doi.org/10.1016/S1074-5521(98)90006-0.
[40]	H.-M. Deng, M.-J. Xiao, Y.-Q. Chai, et al., P3HT-PbS nanocomposites with mimicking enzyme as bi-enhancer for ultrasensitive photocathodic biosensor, Biosens. Bioelectron. 197 (2022) 113806 https://doi.org/10.1016/j.bios.2021.113806.
[41]	R. Ge, S.M. Zhang, H.J. Dai, et al., G-Quadruplex/Hemin-Mediated Polarity-Switchable and Photocurrent-Amplified System for Escherichia coli O157:H7 Detection, J. Agric. Food Chem. 71 (2023) 16807-16814 https://doi.org/10.1021/acs.jafc.3c06052.
[42]	S. Du, X. Pei, Y. Huang, et al., Hemin/G-quadruplex and AuNPs-MoS(2) based novel dual signal amplification strategy for ultrasensitively sandwich-type electrochemical thrombin aptasensor, Bioelectrochemistry 157 (2024) 108635 https://doi.org/10.1016/j.bioelechem.2023.108635.
[43]	X. Xie, X. Cheng, J. Dong, et al., Visual Assay for Methicillin-Resistant Staphylococcus aureus Based on Rolling Circular Amplification Triggering G-Quadruplex/Hemin DNAzyme Proximity Assembly, Anal. Chem. 95 (2023) 3098-3107 https://doi.org/10.1021/acs.analchem.2c05712.
[44]	S. Huang, B. Li, P. Mu, et al., Highly sensitive detection of microRNA-21 by nitrogen-doped carbon dots-based ratio fluorescent probe via nuclease-assisted rolling circle amplification strategy, Anal. Chim. Acta 1273 (2023) 341533 https://doi.org/10.1016/j.aca.2023.341533.
[45]	H. Yang, B. Weng, S. Liu, et al., Acid-improved DNAzyme-based chemiluminescence miRNA assay coupled with enzyme-free concatenated DNA circuit, Biosens. Bioelectron. 204 (2022) 114060 https://doi.org/10.1016/j.bios.2022.114060.
[46]	C.S.V. Rajendrakumar, T. Suryanarayana, A.R. Reddy, DNA helix destabilization by proline and betaine: possible role in the salinity tolerance process, FEBS Lett. 410 (1997) 201-205 https://doi.org/10.1016/S0014-5793(97)00588-7.
[47]	W.A.R.D.Y.K.H.V. Hippel, Betaine can eliminate the base pair composition dependence of DNA melting \| Biochemistry, Biochemistry 32 (1993) 137-144 https://doi.org/10.1021/bi00052a019.
[48]	L. Yijun, S. Jinglu, C. Yongping, et al., MiR-200a acts as an oncogene in colorectal carcinoma by targeting PTEN, Exp. Mol. Pathol. 101 (2016) 308-313 https://doi.org/10.1016/j.yexmp.2016.10.006.