DNAzyme-based probe for circulating microRNA detection in peripheral blood

Abstract

Guoli Shao,1,* Shufeng Ji,1,* Aiguo Wu,1 Cuiping Liu,2 Mengchuan Wang,1 Pusheng Zhang,1 Qingli Jiao,1 Yuzhan Kang2 1Department of General Surgery, 2Department of Critical Care Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People’s Republic of China *These authors contributed equally to this work Background: The recent discovery of microRNAs (miRNAs) and their extracellular presence suggest a potential role of these regulatory molecules in defining the metastatic potential of cancer cells and mediating the cancer–host communication. This study aims to improve the sensitivity of miRNA detection via DNAzyme-based method and enhance the selectivity by using the DNAzyme-based probe to reduce nonspecific amplification. Methods: The miRNA probes were chemically synthesized with a phosphate at the 5' end and purified by polyacrylamide gel electrophoresis. Exosomal RNA from peripheral blood was isolated. Carboxylated magnetic microsphere beads (MBs) were functionalized with streptavidin (SA) according to a previously reported method with some modification. T capture probe-coated SA-MBs (DNA-MBs) were also prepared. The fluorescent spectra were measured using a spectrofluorophotometer. Results: We designed an incomplete DNAzyme probe with two stems and one bubble structure as a recognition element for the specific detection of miRNA with high sensitivity. The background effects were decreased with increase of the added of DNA-MBs and capturing times. Therefore, 20 minutes was selected as the optimal concentration in the current study. The fluorescence intensity increases as the hybridization time changed and reached a constant level at 40 minutes, and 1 µM is the optimum signal probe concentration for self-assembled DNA concatemers formation. In the presence of miRNA, the fluorescence of the solution increased with increasing miRNA concentration. There is no obvious fluorescence in the presence of 10 mM of other nontarget DNA. Conclusion: A simple, rapid method with high performance has been constructed based on identified circulating miRNA signatures using miRNA-induced DNAzyme. This assay is simple, inexpensive, and sensitive, enabling quantitative detection of as low as 10 fM miRNA. Keywords: DNAzyme, miRNAs, breast cancer, detection