Abstract
Highly sensitive detection of bone morphogenetic protein 6 (BMP6) mRNA is essential to monitor bone regeneration in the regenerating defects. In this work, we proposed a quantitative approach based on two-stage nicking enzyme signal amplification (NESA) and DNAzyme amplification for highly sensitive detection of BMP6 mRNA. The two-stage NESA involves two templates and two-stage amplification reactions under isothermal conditions. The first template contains two repeat sequences that could hybridize to the target RNA, triggering an exponential amplification. The amplified product was a short single-stranded DNA with the same sequence as the target RNA. The single-stranded DNA can trigger another linear NESA and produces a large amount of horseradish peroxidase (HRP)-mimicking G-quadruplex DNAzyme. This proposed assay showed a quantitative analysis of BMP6 mRNA in a wide range from 1 fM to 100 nM with a detection limit of 0.01 fM.
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References
B. R. Cullen, Mol. Cell, 2004, 16, 861.
J. A. Lednicky, Arch. Pathol. Lab. Med., 2003, 127, 30.
W. W. Fischer, Nature, 2008, 455, 1051.
K. Hirota, T. Miyoshi, K. Kugou, C. S. Hoffman, T. Shibata, and K. Ohta, Nature, 2008, 456, 130.
A. Valoczi, C. Hornyik, N. Varga, J. Burgyan, S. Kauppinen, and Z. Havelda, Nucleic Acids Res., 2004, 32, e175.
J. T. Pena, C. Sohn-Lee, S. H. Rouhanifard, J. Ludwig, M. Hafner, A. Mihailovic, C. Lim, D. Holoch, P. Berninger, M. Zavolan, and T. Tuschl, Nat. Methods, 2009, 6, 139.
L. P. Lim, N. C. Lau, P. Garrett-Engele, A. Grimson, J. M. Schelter, J. Castle, D. P. Bartel, P. S. Linsley, and J. M. Johnson, Nature, 2005, 433, 769.
J. Baner, M. Nilsson, M. Mendel-Hartvig, and U. Landegren, Nucleic Acids Res., 1998, 26, 5073.
R. K. Saiki, D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich, Sciences (N.Y.), 1988, 239, 487.
R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim, Sciences (N.Y.), 1985, 230, 1350.
B. C. Yin, Y. Q. Liu, and B. C. Ye, Anal. Chem., 2013, 85, 11487.
J. Li, B. Yao, H. Huang, Z. Wang, C. Sun, Y. Fan, Q. Chang, S. Li, X. Wang, and J. Xi, Anal. Chem., 2009, 81, 5446.
Y. Zhang and C. Y. Zhang, Anal. Chem., 2012, 84, 224.
G. L. Wang and C. Y. Zhang, Anal. Chem., 2012, 84, 7037.
C. Li, Z. Li, H. Jia, and J. Yan, Chem. Commun. (Cambridge, U. K.), 2011, 47, 2595.
Y. Cheng, X. Zhang, Z. Li, X. Jiao, Y. Wang, and Y. Zhang, Angew. Chem., Int. Ed. Engl., 2009, 48, 3268.
X. P. Wang, B. C. Yin, P. Wang, and B. C. Ye, Biosens. Bioelectron., 2013, 42, 131.
P. J. Asiello and A. J. Baeumner, Lab Chip, 2011, 11, 1420.
H. Zhang, F. Li, B. Dever, X.-F. Li, and X. C. Le, Chem. R ev. , 2012, 113, 2812.
L. Qiu, C. Wu, M. You, D. Han, T. Chen, G. Zhu, J. Jiang, R. Yu, and W. Tan, J. Am. Chem. Soc., 2013, 135, 12952.
Y. Li, X. Li, X. Ji, and X. Li, Biosens. Bioelectron., 2011, 26, 4095.
J. Liu, C. Y. Lu, H. Zhou, J. J. Xu, Z. H. Wang, and H. Y. Chen, Chem. Commun. (Cambridge, U. K.), 2013, 49, 6602.
J. Li, Q. H. Yao, H. E. Fu, X. L. Zhang, and H. H. Yang, Talanta, 2011, 85, 91
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Hu, Z., Zhou, Y., Xie, X. et al. Two-stage Nicking Enzyme Signal Amplification Combined with DNAzyme Amplification for the Detection of Bone Morphogenetic Protein 6 mRNA. ANAL. SCI. 30, 1039–1044 (2014). https://doi.org/10.2116/analsci.30.1039
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DOI: https://doi.org/10.2116/analsci.30.1039