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Isothermal exponential amplification reactions triggered by circular templates (cEXPAR) targeting miRNA

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Abstract

Background

Isothermal exponential amplification reaction (EXPAR) is an emerging amplification technique that is most frequently used to amplify microRNA (miRNA). However, EXPAR also exhibits non-specific background amplification in the absence of the targeted sequence, which limits the attainable assay sensitivity of EXPAR.

Methods and results

A novel modified isothermal EXPAR based on circular amplification templates (cEXPAR) was developed in this study. The circular template consists of two same linear fragments that complement the target sequence, and these two linear fragments are separated by two nicking agent recognition sequences (NARS). Compared with the linear structure template, this circular template allows DNA or RNA fragments to be randomly paired with two repeated sequences and can be successfully amplified. This reaction system developed in this study could rapidly synthesize short oligonucleotide fragments (12-22 bp) through simultaneous nicking and displacement reactions. Highly sensitive chain reactions can be specifically triggered by as low as a single copy of target molecule, and non-specific amplification can be effectively eliminated in this optimized system. Moreover, the proposed approach applied to miRNA test can discriminate single-nucleotide variations between miRNAs.

Conclusion

The newly developed cEXPAR assay provides a useful alternative tool for rapid, sensitive, and highly specific detection of miRNAs.

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References

  1. Zhao L, Liang X, Wang L, Zhang X (2022) The role of miRNA in Ovarian Cancer: an overview. Reprod Sci 29:2760–2767. https://doi.org/10.1007/s43032-021-00717-w

    Article  CAS  PubMed  Google Scholar 

  2. Hill M, Tran N (2021) miRNA interplay: mechanisms and consequences in cancer. Dis Model Mech 14. https://doi.org/10.1242/dmm.047662

  3. Cullen BR (2013) MicroRNAs as mediators of viral evasion of the immune system. Nat Immunol 14:205–210. https://doi.org/10.1038/ni.2537

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lin S, Gregory RI (2015) MicroRNA biogenesis pathways in cancer. Nat Rev Cancer 15:321–333. https://doi.org/10.1038/nrc3932

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Panda M, Kalita E, Singh S, Kumar K, Rao A, Prajapati VK (2022) MiRNA-SARS-CoV-2 dialogue and prospective anti-COVID-19 therapies. Life Sci 305:120761. https://doi.org/10.1016/j.lfs.2022.120761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Rupaimoole R, Slack FJ (2017) MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat Rev Drug Discovery 16:203–222. https://doi.org/10.1038/nrd.2016.246

    Article  CAS  PubMed  Google Scholar 

  7. Ho PTB, Clark IM, Le LTT (2022) MicroRNA-Based diagnosis and therapy. Int J Mol Sci 23. https://doi.org/10.3390/ijms23137167

  8. Wu Y, Yuan W, Ding H, Wu X (2022) Serum exosomal miRNA from endometriosis patients correlates with disease severity. Arch Gynecol Obstet 305:117–127. https://doi.org/10.1007/s00404-021-06227-z

    Article  CAS  PubMed  Google Scholar 

  9. Wang J, Wen J, Yan H (2021) Recent applications of Carbon Nanomaterials for microRNA Electrochemical sensing. Chem Asian J 16:114–128. https://doi.org/10.1002/asia.202001260

    Article  CAS  PubMed  Google Scholar 

  10. Ban E, Song EJ (2022) Considerations and suggestions for the Reliable analysis of miRNA in plasma using qRT-PCR. Genes. 13. https://doi.org/10.3390/genes13020328

  11. Válóczi A, Hornyik C, Varga N, Burgyán J, Kauppinen S, Havelda Z (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 32:e175. https://doi.org/10.1093/nar/gnh171

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hurley J, Roberts D, Bond A, Keys D, Chen C (2012) Stem-loop RT-qPCR for microRNA expression profiling. Methods in molecular biology. (Clifton N J) 822:33–52. https://doi.org/10.1007/978-1-61779-427-8_3

    Article  CAS  Google Scholar 

  13. Smith I, Halpin K, Warrilow D, Smith GA (2001) Development of a fluorogenic RT-PCR assay (TaqMan) for the detection of Hendra virus. J Virol Methods 98:33–40. https://doi.org/10.1016/S0166-0934(01)00354-8

    Article  CAS  PubMed  Google Scholar 

  14. Luo X, Zhang J, Wang H, Du Y, Yang L, Zheng F, Ma D (2011) PolyA RT-PCR-based quantification of microRNA by using universal TaqMan probe. Biotechnol Lett 34:627–633. https://doi.org/10.1007/s10529-011-0813-3

    Article  CAS  PubMed  Google Scholar 

  15. Erdogan I, Cosacak M, Nalbant A, Akgül B (2018) Deep sequencing reveals two jurkat subpopulations with distinct miRNA profiles during camptothecin-induced apoptosis. TURKISH J BIOLOGY 42. https://doi.org/10.3906/biy-1710-62

  16. Wei R, Zhang R, Li H, Li H, Zhang S, Xie Y, Shen L, Chen F (2018) MiR-29 targets PUMA to suppress oxygen and glucose Deprivation/Reperfusion (OGD/R)-induced cell death in hippocampal neurons. Curr Neurovasc Res 15:47–54. https://doi.org/10.2174/1567202615666180403170902

    Article  CAS  PubMed  Google Scholar 

  17. Moltzahn F, Olshen AB, Baehner L, Peek A, Fong L, Stöppler H, Simko J, Hilton JF, Carroll P, Blelloch R (2011) Microfluidic-based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in the sera of prostate cancer patients. Cancer Res 71:550–560. https://doi.org/10.1158/0008-5472.Can-10-1229

    Article  CAS  PubMed  Google Scholar 

  18. Bettazzi F, Hamid-Asl E, Esposito C, Quintavalle C, Formisano N, Laschi S, Catuogno S, Iaboni M, Marrazza G, Mascini M, Cerchia L, de Franciscis V, Condorelli G, Palchetti I (2012) Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay. Analytical and bioanalytical chemistry 405. https://doi.org/10.1007/s00216-012-6476-7

  19. Li T, Wu X, Tao G, Yin H, Zhang J, Liu F, Li N (2018) A simple and non-amplification platform for femtomolar DNA and microRNA detection by combining automatic gold nanoparticle enumeration with target-induced strand-displacement. Biosens Bioelectron 105:137–142. https://doi.org/10.1016/j.bios.2018.01.034

    Article  CAS  PubMed  Google Scholar 

  20. Yin P, Choi HMT, Calvert CR, Pierce NA (2008) Programming biomolecular self-assembly pathways. Nature 451:318–322. https://doi.org/10.1038/nature06451

    Article  CAS  PubMed  Google Scholar 

  21. Dirks RM, Pierce NA (2004) Triggered amplification by hybridization chain reaction. Proc Natl Acad Sci U S A 101:15275–15278. https://doi.org/10.1073/pnas.0407024101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Li C, Li Z, Jia H, Yan J (2011) One-step ultrasensitive detection of microRNAs with loop-mediated isothermal amplification (LAMP). Chem Commun (Camb) 47:2595–2597. https://doi.org/10.1039/c0cc03957h

    Article  CAS  PubMed  Google Scholar 

  23. Zhang J, Li C, Zhi X, Ramón GA, Liu Y, Zhang C, Pan F, Cui D (2016) Hairpin DNA-Templated silver nanoclusters as novel beacons in strand displacement amplification for MicroRNA detection. Anal Chem 88:1294–1302. https://doi.org/10.1021/acs.analchem.5b03729

    Article  CAS  PubMed  Google Scholar 

  24. Jonstrup SP, Koch J, Kjems J (2006) A microRNA detection system based on padlock probes and rolling circle amplification. RNA 12:1747–1752. https://doi.org/10.1261/rna.110706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yin BC, Liu YQ, Ye BC (2012) One-step, multiplexed fluorescence detection of microRNAs based on duplex-specific nuclease signal amplification. J Am Chem Soc 134:5064–5067. https://doi.org/10.1021/ja300721s

    Article  CAS  PubMed  Google Scholar 

  26. Van Ness J, Van Ness LK, Galas DJ (2003) Isothermal reactions for the amplification of oligonucleotides. Proceedings. Natl Acad Sci United States Am 100:4504–4509. https://doi.org/10.1073/pnas.0730811100

    Article  CAS  Google Scholar 

  27. Jia H, Li Z, Liu C, Cheng Y (2010) Ultrasensitive detection of microRNAs by exponential isothermal amplification. Angew Chem Int Ed Engl 49:5498–5501. https://doi.org/10.1002/anie.201001375

    Article  CAS  PubMed  Google Scholar 

  28. Yu Y, Chen Z, Shi L, Yang F, Pan J, Zhang B, Sun D (2014) Ultrasensitive electrochemical detection of microRNA based on an arched probe mediated isothermal exponential amplification. Anal Chem 86:8200–8205. https://doi.org/10.1021/ac501505a

    Article  CAS  PubMed  Google Scholar 

  29. Xiang M, Zeng Y, Yang R, Xu H, Chen Z, Zhong J, Xie H, Xu Y, Zeng X (2014) U6 is not a suitable endogenous control for the quantification of circulating microRNAs. Biochem Biophys Res Commun 454:210–214. https://doi.org/10.1016/j.bbrc.2014.10.064

    Article  CAS  PubMed  Google Scholar 

  30. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods (San Diego Calif) 25(4):402–408. https://doi.org/10.1006/meth.2001.1262

    Article  CAS  PubMed  Google Scholar 

  31. Huang JF, Zhao N, Xu HQ, Xia H, Wei K, Fu WL, Huang Q (2016) Sensitive and specific detection of miRNA using an isothermal exponential amplification method using fluorescence-labeled LNA/DNA chimera primers. Anal Bioanal Chem 408:7437–7446. https://doi.org/10.1007/s00216-016-9829-9

    Article  CAS  PubMed  Google Scholar 

  32. Wang GL, Zhang CY (2012) Sensitive detection of microRNAs with hairpin probe-based circular exponential amplification assay. Anal Chem 84:7037–7042. https://doi.org/10.1021/ac3012544

    Article  CAS  PubMed  Google Scholar 

  33. Duan R, Zuo X, Wang S, Quan X, Chen D, Chen Z, Jiang L, Fan C, Xia F (2014) Quadratic isothermal amplification for the detection of microRNA. Nat Protoc 9:597–607. https://doi.org/10.1038/nprot.2014.036

    Article  CAS  PubMed  Google Scholar 

  34. Tan E, Erwin B, Dames S, Ferguson T, Buechel M, Irvine B, Voelkerding K, Niemz A (2008) Specific versus nonspecific isothermal DNA amplification through Thermophilic polymerase and nicking enzyme activities †. Biochemistry 47:9987–9999. https://doi.org/10.1021/bi800746p

    Article  CAS  PubMed  Google Scholar 

  35. Mok E, Wee E, Wang Y, Trau M (2016) Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction. Sci Rep 6:37837. https://doi.org/10.1038/srep37837

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ma L, Teruya-Feldstein J, Weinberg RA (2007) Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449:682–688. https://doi.org/10.1038/nature06174

    Article  CAS  PubMed  Google Scholar 

  37. Hannafon BN, Carpenter KJ, Berry WL, Janknecht R, Dooley WC, Ding WQ (2015) Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA). Mol Cancer 14:133. https://doi.org/10.1186/s12943-015-0400-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by [National Natural Science Foundation of China] (Grant numbers [81572066] and [32001084]) and [Natural Science Foundation of Chongqing] (Grant numbers [cstc2020jcyj-msxmX0173]).

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  1. The authors Xue-mei Qu and Xiao-dong Ren have contributed equally to this work

Authors and Affiliations

  1. Department of Laboratory Medicine, Daping Hospital, Army Medical University, 400042, Chongqing, P.R. China

    Xue-mei Qu, Xiao-dong Ren, Ning Su, Xian-ge Sun, Shao-li Deng, Wei-ping Lu & Qing Huang

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Contributions

QH conceived and designed the study. XMQ, XDR and NS, performed the experiments and interpreted the results. XDR and XMQ wrote the manuscript. XGS edited the figures and references in the manuscript. XGS, SLD and WPL assisted in conducting the experiments and analyzed the data. All authors read and approved the final version of the manuscript.

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Correspondence to Qing Huang.

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Qu, Xm., Ren, Xd., Su, N. et al. Isothermal exponential amplification reactions triggered by circular templates (cEXPAR) targeting miRNA. Mol Biol Rep 50, 3653–3659 (2023). https://doi.org/10.1007/s11033-023-08291-x

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