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Nicking-enhanced rolling circle amplification for sensitive fluorescent detection of cancer-related microRNAs

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Abstract

In this study, a biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) was proposed for the highly sensitive detection of cancer-related let-7a microRNA (miRNA). The sensing system consists of a padlock probe (PP), which contains a target recognition sequence and two binding sites for nicking endonuclease (NEase), and molecular beacon (MB) as reporting molecule. Upon hybridization with let-7a, the PP can be circularized by ligase. Then, the miRNA acted as polymerization primer to initiate rolling circle amplification (RCA). With the assistance of NEase, RCA products can be nicked on the cyclized PP and are displaced during the subsequent duplication process, generating numerous nicked fragments (NFs). These NFs not only induce another RCA reaction but also open the molecular beacons (MBs) via hybridization, leading to significantly amplified fluorescence signal. Under the optimized conditions, this method exhibits high sensitivity toward target miRNA let-7a with a detection limit of as low as 10 pM, a dynamic range of three orders of magnitude is achieved, and its family member is easily distinguished even with only one mismatched base. Meanwhile, it displays good recovery and satisfactory reproducibility in fetal bovine serum (FBS). Therefore, these merits endow the newly proposed N-RCA strategy with powerful implications for miRNA detection.

A biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) for the highly sensitive detection of cancer-related let-7a microRNA

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References

  1. Friedman RC, Farh KK-H, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 2009;19:92–105.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Liang Y, Ridzon D, Wong L, Chen C. Characterization of microRNA expression profiles in normal human tissues. BMC Genomics. 2007;8:166.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, et al. RAS is regulated by the let-7 microRNA family. Cell. 2005;120:635–47.

    Article  CAS  PubMed  Google Scholar 

  4. Van Kouwenhove M, Kedde M, Agami R. MicroRNA regulation by RNA-binding proteins and its implications for cancer. Nat Rev Cancer. 2011;11:644–56.

    Article  CAS  PubMed  Google Scholar 

  5. Belgardt B-F, Ahmed K, Spranger M, Latreille M, Denzler R, Kondratiuk N, et al. The microRNA-200 family regulates pancreatic beta cell survival in type 2 diabetes. Nat Med. 2015;21:619–27.

    Article  CAS  PubMed  Google Scholar 

  6. Seronde M-F, Vausort M, Gayat E, Goretti E, Ng LL, Squire IB, et al. Circulating microRNAs and outcome in patients with acute heart failure. PLoS One. 2015;10:e0142237.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jeanson-Leh L, Lameth J, Krimi S, Buisset J, Amor F, Le Guiner C, et al. Serum profiling identifies novel muscle miRNA and cardiomyopathy-related miRNA biomarkers in Golden Retriever muscular dystrophy dogs and Duchenne muscular dystrophy patients. Am J Pathol. 2014;184:2885–98.

    Article  CAS  PubMed  Google Scholar 

  8. Li J, Tan S, Kooger R, Zhang C, Zhang Y. MicroRNAs as novel biological targets for detection and regulation. Chem Soc Rev. 2014;43:506–17.

    Article  CAS  PubMed  Google Scholar 

  9. Brueckner B, Stresemann C, Kuner R, Mund C, Musch T, Meister M, et al. The human let-7a-3 locus contains an epigenetically regulated microRNA gene with oncogenic function. Cancer Res. 2007;67:1419–23.

    Article  CAS  PubMed  Google Scholar 

  10. Válóczi A, Hornyik C, Varga N, Burgyán J, Kauppinen S, Havelda Z. Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res. 2004;32:e175.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cissell KA, Shrestha S, Deo SK. MicroRNA detection: challenges for the analytical chemist. Anal Chem. 2007;79:4754–61.

    Article  CAS  Google Scholar 

  12. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, et al. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Res. 2005;33:e179.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Benes V, Castoldi M. Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods. 2010;50:244–9.

    Article  CAS  PubMed  Google Scholar 

  14. Zhang J, Li Z, Wang H, Wang Y, Jia H, Yan J. Ultrasensitive quantification of mature microRNAs by real-time PCR based on ligation of a ribonucleotide-modified DNA probe. Chem Commun. 2011;47:9465–7.

    Article  CAS  Google Scholar 

  15. Thomson JM, Parker J, Perou CM, Hammond SM. A custom microarray platform for analysis of microRNA gene expression. Nat Methods. 2004;1:47–53.

    Article  CAS  PubMed  Google Scholar 

  16. Lee JM, Jung Y. Two-temperature hybridization for microarray detection of label-free microRNAs with attomole detection and superior specificity. Angew Chem Int Edit. 2011;50:12487–90.

    Article  CAS  Google Scholar 

  17. Harcourt EM, Kool ET. Amplified microRNA detection by templated chemistry. Nucleic Acids Res. 2012;40:e65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Baker MB, Bao G, Searles CD. In vitro quantification of specific microRNA using molecular beacons. Nucleic Acids Res. 2011;40:e13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Huang J, Su X, Li Z. Enzyme-free and amplified fluorescence DNA detection using bimolecular beacons. Anal Chem. 2012;84:5939–43.

    Article  CAS  PubMed  Google Scholar 

  20. Xu H, Zhang R, Li F, Zhou Y, Peng T, Wang X, et al. Double-hairpin molecular-beacon-based amplification detection for gene diagnosis linked to cancer. Anal Bioanal Chem. 2016;408:6181–8.

    Article  CAS  PubMed  Google Scholar 

  21. Xu J, Wu ZS, Wang Z, Le J, Zheng T, Jia L. Autonomous assembly of ordered metastable DNA nanoarchitecture and in situ visualizing of intracellular microRNAs. Biomaterials. 2017;120:57–65.

    Article  CAS  PubMed  Google Scholar 

  22. Xu J, Li H, Wu ZS, Qian J, Xue C, Jia L. Double-stem hairpin probe and ultrasensitive colorimetric detection of cancer-related nucleic acids. Theranostics. 2016;6:318–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Xu H, Xue C, Zhang R, Chen Y, Li F, Shen Z, et al. Exponential rolling circle amplification and its sensing application for highly sensitive DNA detection of tumor suppressor gene. Sensors Actuators B Chem. 2017;243:1240–7.

    Article  CAS  Google Scholar 

  24. Li F, Zhao H, Wang Z-Y, Wu Z-S, Yang Z, Li C-C, et al. Single palindromic molecular beacon-based amplification for genetic analysis of cancers. Biosens Bioelectron. 2017;91:692–8.

    Article  CAS  PubMed  Google Scholar 

  25. Shen Z-F, Li F, Jiang Y-F, Chen C, Xu H, Li C-C, et al. Palindromic molecule beacon-based cascade amplification for colorimetric detection of cancer genes. Anal Chem. 2018;90:3335–40.

    Article  CAS  PubMed  Google Scholar 

  26. Zhou H, Xie SJ, Zhang SB, Shen GL, Yu RQ, Wu ZS. Isothermal amplification system based on template-dependent extension. Chem Commun. 2013;49:2448–50.

    Article  CAS  Google Scholar 

  27. Bi S, Cui Y, Dong Y, Zhang N. Target-induced self-assembly of DNA nanomachine on magnetic particle for multi-amplified biosensing of nucleic acid, protein, and cancer cell. Biosens Bioelectron. 2014;53:207–13.

    Article  CAS  PubMed  Google Scholar 

  28. Xu H, Wu D, Zhang Y, Shi H, Ouyang C, Li F, et al. RCA-enhanced multifunctional molecule beacon-based strand-displacement amplification for sensitive microRNA detection. Sensors Actuators B Chem. 2018;258:470–7.

    Article  CAS  Google Scholar 

  29. Wang Z-Y, Li F, Zhang Y, Zhao H, Xu H, Wu Z-S, et al. Sensitive detection of cancer gene based on a nicking-mediated RCA of circular DNA nanomachine. Sensors Actuators B Chem. 2017;251:692–8.

    Article  CAS  Google Scholar 

  30. Ge J, Dong Z-Z, Bai D-M, Zhang L, Hu Y-L, Ji D-Y, et al. A novel label-free fluorescent molecular beacon for the detection of 3′–5′ exonuclease enzymatic activity using DNA-templated copper nanoclusters. New J Chem. 2017;41:9718–23.

    Article  CAS  Google Scholar 

  31. Huang Z-M, Cai Q-Y, Ding D-C, Ge J, Hu Y-L, Yang J, et al. A facile label-free colorimetric method for highly sensitive glutathione detection by using manganese dioxide nanosheets. Sensors Actuators B Chem. 2017;242:355–61.

    Article  CAS  Google Scholar 

  32. Luo C, Tang H, Cheng W, Yan L, Zhang D, Ju H, et al. A sensitive electrochemical DNA biosensor for specific detection of Enterobacteriaceae bacteria by exonuclease III-assisted signal amplification. Biosens Bioelectron. 2013;48:132–7.

    Article  CAS  PubMed  Google Scholar 

  33. Wang M, Fu Z, Li B, Zhou Y, Yin H, Ai S. One-step, ultrasensitive, and electrochemical assay of microRNAs based on T7 exonuclease assisted cyclic enzymatic amplification. Anal Chem. 2014;86:5606–10.

    Article  CAS  PubMed  Google Scholar 

  34. Wu Z-S, Zhou H, Zhang S, Shen G, Yu R. Electrochemical aptameric recognition system for a sensitive protein assay based on specific target binding-induced rolling circle amplification. Anal Chem. 2010;82:2282–9.

    Article  CAS  PubMed  Google Scholar 

  35. Chapin SC, Doyle PS. Ultrasensitive multiplexed microRNA quantification on encoded gel microparticles using rolling circle amplification. Anal Chem. 2011;83:7179–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nilsson M, Antson DO, Barbany G, Landegren U. RNA-templated DNA ligation for transcript analysis. Nucleic Acids Res. 2001;29:578–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Qiu L, Qiu L, Zhou H, Wu Z, Shen G, Yu R. Sensitive and selective electrochemical DNA sensor for the analysis of cancer-related single nucleotide polymorphism. New J Chem. 2014;38:4711–5.

    Article  CAS  Google Scholar 

  38. Wu ZS, Jiang JH, Shen GL, Yu RQ. Highly sensitive DNA detection and point mutation identification: an electrochemical approach based on the combined use of ligase and reverse molecular beacon. Hum Mutat. 2010;28:630–7.

    Article  CAS  Google Scholar 

  39. Yu J, 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;135:7430–3.

    Article  CAS  Google Scholar 

  40. Liu H, Li L, Duan L, Wang X, Xie Y, Tong L, et al. High specific and ultrasensitive isothermal detection of microRNA by padlock probe-based exponential rolling circle amplification. Anal Chem. 2013;85:7941–7.

    Article  CAS  PubMed  Google Scholar 

  41. Feng K, Qiu LP, Yang Y, Wu ZS, Shen GL, Yu RQ. Label-free optical bifunctional oligonucleotide probe for homogeneous amplification detection of disease markers. Biosens Bioelectron. 2011;29:66–75.

    Article  CAS  PubMed  Google Scholar 

  42. Dong H, Ma J, Wang J, Wu ZS, Sinko PJ, Jia L. A biofunctional molecular beacon for detecting single base mutations in cancer cells. Mol Ther Nucl Acids. 2016;5:e302.

    Article  CAS  Google Scholar 

  43. Zhang S, Wu Z, Shen G, Yu R. A label-free strategy for SNP detection with high fidelity and sensitivity based on ligation-rolling circle amplification and intercalating of methylene blue. Biosens Bioelectron. 2009;24:3201–7.

    Article  CAS  PubMed  Google Scholar 

  44. Deng R, Tang L, Tian Q, Wang Y, Lin L, Li J. Toehold-initiated rolling circle amplification for visualizing individual microRNAs in situ in single cells. Angew Chem Int Edit. 2014;53:2389–93.

    Article  CAS  Google Scholar 

  45. Simms D, Cizdziel PE, Chomczynski P. TRIzol: a new reagent for optimal single-step isolation of RNA. Focus. 1993;15:532–5.

    Google Scholar 

  46. Hu J, Wen C-Y, Zhang Z-L, Xie M, Hu J, Wu M, et al. Optically encoded multifunctional nanospheres for one-pot separation and detection of multiplex DNA sequences. Anal Chem. 2013;85:11929–35.

    Article  CAS  PubMed  Google Scholar 

  47. Cheng Y, Zhang X, Li Z, Jiao X, Wang Y, Zhang Y. Highly sensitive determination of microRNA using target-primed and branched rolling-circle amplification. Angew Chem Int Edit. 2009;121:3318–22.

    Article  Google Scholar 

  48. Hikichi M, Kidokoro M, Haraguchi T, Iba H, Shida H, Tahara H, et al. MicroRNA regulation of glycoprotein B5R in oncolytic vaccinia virus reduces viral pathogenicity without impairing its antitumor efficacy. Mol Ther. 2011;19:1107–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Chen, Lijia, Xing, Yuan, Kehui, Wang, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 2008;18:997–1006.

    Article  CAS  PubMed  Google Scholar 

  50. Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet. 2009;10:704–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by the National Natural Science Foundation of China (NSFC) (grant no: 21775024) and Zhejiang Province Natural Science Foundation of China (LY16C07002).

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Author notes

  1. Zhihua Gao and Chengwei Wu contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Laboratory Medicine, Ministry of Education of China, Zhejiang Provincial Key Laboratory of Medicine Genetics, School of Laboratory Medicine and Life Sciences, Institute of Functional Nucleic Acids and Personalized Cancer Theranostics, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China

    Zhihua Gao, Chengwei Wu, Sha Lv, Cong Wang, Nan Zhang, Shuai Xiao, Ying Han, Yan Zhang, Feng Li, Jianxin Lyu & Zhifa Shen

  2. Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350002, Fujian, China

    Shuai Xiao, Ying Han, Huo Xu & Feng Li

  3. Department of Laboratory Medicine, People’s Hospital of Hangzhou Medical College, Hangzhou, 310053, Zhejiang, China

    Jianxin Lyu

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  1. Zhihua Gao
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Corresponding authors

Correspondence to Feng Li, Jianxin Lyu or Zhifa Shen.

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This study was approved by the ethical review committee of The First Affiliated Hospital of Wenzhou. Written informed consent was obtained by the volunteer the human serum sample was obtained from.

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The authors declare that they have no conflict of interest.

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Gao, Z., Wu, C., Lv, S. et al. Nicking-enhanced rolling circle amplification for sensitive fluorescent detection of cancer-related microRNAs. Anal Bioanal Chem 410, 6819–6826 (2018). https://doi.org/10.1007/s00216-018-1277-2

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