Ultrasensitive label-free detection of miRNA with asymmetric hairpin probe, exonuclease I and SYBR Green I

Abstract

Detection of miRNAs presents a particular challenge because of their limited size, high sequence homology and greatly various expression level. In this work, an ultrasensitive, label-free and isothermal miRNA detection was developed based on asymmertric hairpin probe, exonuclease I(Exo I) and SYBR Green I. The method employed asymmetric hairpin probe to perform cycled polymerization and Exo I to reduce background signal. In the presence of the target miRNA, the target triggers probe-mediated cycled polymerization reactions to generate lots of dsDNA products. The dsDNA product effectively prevents itself from being degraded by Exo I and permitted the insertion of more fluorescence dye into it to enlarge the fluorescence signal. In the absence of the target miRNA, there was no probe-mediated polymerization reaction, and the probe was digested by Exo I added, which minimized the intercalation of fluorescence dye to reduce the background signal. The combination of the probe-mediated cycled polymerization with the Exo I-assisted background reduction allows us to achieve a detection limit of 5×10⁻¹⁸ mol/L. Owing to its ultrasensitivity, excellent specificity, convenience and low-cost, this method might hold out great promise in miRNA detection.

Keywords

Ultrasensitive / miRNA / Asymmetric hairpin probe / Exonuclease I

References

[1] Ambros V. Nature, 2004, 431: 350.

[2] Friedman R C, Farh K K H, Burge C B, Bartel D P. Genome Res., 2009, 19: 92.

[3] Zha Y, He S M, Liu C N, Ru S W, Zha H T, Yang Z, Yang P C, Yuan X Y, Sun S W, Bu D B, Huan J F, Skogerbo G, Chen R S. Trends Gene, 2008, 24: 323.

[4] Bartel D P. Cell, 2009, 136: 215.

[5] Schickel R, Boyerinas B, Park S M, Peter M E. Oncogene, 2008, 27: 5959.

[6] Cissell K A, Shrestha S, Deo S K. Anal. Chem., 2007, 79: 4754.

[7] Lei H, Zou D, Li Z. PLoS One, 2013, 8: e60369.

[8] Volkomorov V, Grigoryeva E, Krasnov G, Litviakov N, Tsyganov M. Exp. Oncol., 2013, 35: 2.

[9] Valoczi A, Hornyik C, Varga N, Burgyán J, Kauppinen S, Havelda Z. Nucleic Acids Res., 2004, 32: e175.

[10] Ichikawa M, Akiyama H. Methods Mol. Biol., 2013, 1024: 247.

[11] Jacques C, Guillotin D, Fontaine J F, Franc B, Mirebeau P D, Fleury A, Malthiery Y, Savagner F. Clin. Endocrinol. Metab., 2013, 98: 981.

[12] Cao S, Chen S J. Nucleic Acids Res., 2012, 40: 4681.

[13] Huang J, Wu Y R, Chen Y, Zhu Z, Yang X, Yang C, Wang K, Tan W. Angew. Chem. Int. Ed., 2011, 50: 401.

[14] Neubacher S, Arenz C. ChemBioChem., 2009, 10: 1289.

[15] Murakami T, Sumaoka J, Komiyama M. Nucleic Acids Res., 2009, 37: e19.

[16] Harcourt E M, Kool E T. Nucleic Acids Res., 2012, 40: e65.

[17] Caruana D J, Heller A. J. Am. Chem. Soc., 1999, 121: 769.

[18] Lie P C, Liu J, Fang Z Y, Dun B Y, Zeng L W. Chem. Commun., 2012, 48: 236.

[19] Guo Q P, Yang X H, Wang K H, Li W, Tang H, Li H. Nucleic Acids Res., 2009, 37: e20.

[20] Su H Y, Long J B, Guo Q P, Meng X C, Tan Y J, Cai Q Y, Chen Z, Meng X X. Talanta, 2013, 116: 330.

[21] Su H Y, Meng X C, Guo Q P, Tan Y J, Cai Q Y, Qin H T, Meng X X. Biosens. Bioelectron., 2014, 52: 417.

[22] Zhou Y T, Huang Q, Gao J M, Lu J X, Shen X Z, Fan C H. Nucleic Acids Res., 2010, 38: e156.

[23] Joshi G K, Deitz M S, Johnson M, Mali S, Korc M, Sardar R. Nano Lett., 2014, 14: 6955.

[24] Yang C, Dou B, Shi K, Chai Y, Xiang Y, Yuan R. Anal. Chem., 2014, 86: 11913.

[25] Zhu G, Liang L, Zhang C Y. Anal. Chem., 2014, 86: 11410.

[26] Jiang B Y, Wang M, Li C, Xie J Q. Biosens. Bioelectron., 2013, 43: 289.

[27] Zheng D M, Zou R X, Lou X H. Anal. Chem., 2012, 84: 3554.

[28] Murakami T, Sumaoka J, Komiyama M. Nucleic Acids Res., 2009, 37: e19.