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Highly specific quantification of microRNA by coupling probe-rolling circle amplification and Förster resonance energy transfer

Significance Statement

The first microRNA (miRNA) was discovered in C. elegans in 1993. Subsequently, miRNAs have been identified as a class of small non-coding RNAs containing around 21 nucleotides and virtually exist in all mammalian cells. Currently, miRNAs have been regarded as a class of potential diagnostic markers or therapeutic targets for cancer, cardiovascular and others diseases.

The short length, the similarity of nucleotide sequence and extremely low level in living cells have seriously limited the detection of miRNAs with ultimate sensitivity, high specificity and superior accuracy. Up to date, such a limitation has not been adequately overcome by reported methods. In this paper, the authors presented a novel approach (pRCA-FRET) for quantitative detection of miRNA based on a sequential combination of padlock probe-rolling circle amplification (p-RCA) and förster resonance energy transfer (FRET). First, the padlock probe is specifically ligated and circularized with the target miRNA to produce a long ssDNA by p-RCA in the presence of a DNA polymerase. In this step, p-RCA can superiorly distinguish the mismatch between miRNA and padlock probe and increase the specificity towards target miRNAs. Then, FRET probes labeled with florescent groups Cy3 and Cy5 are used to hybridize the ssDNA from p-RCA. After the excitation of the donor fluorophore Cy3, detectable readouts from the fluorescence emission of Cy5 are generated by FRET. Consequently, the target miRNA can be quantified by analyzing the intensity of fluorescent emission. Compared to previous methods, the sensitivity and operational simplicity have obviously been improved in the step of FRET without complex PCR amplification, accompanying a further enhancement of specificity for miRNA detection.

Using pRCA-FRET, the detection limit of miRNA quantification is markedly reduced from fM level to 103 aM, and remarkable specificity is exemplified to differentiate single-base mismatch between target miRNA and similar miRNAs. Accordingly, this pRCA-FRET has the potential to quantify low amount of miRNA with excellent sensitivity and specificity for the exploration of the biological functions of miRNAs and their clinical applications.  

quantification of microRNA by coupling probe-rolling circle amplification and Förster resonance energy transfer-global medical discovery

About The Author

Prof. Dr. Yijun Chen received a B.S. degree in pharmaceutical science at China Pharmaceutical University, Nanjing, China, in 1982 and completed his Ph.D. degree in medicinal and natural products chemistry at University of Iowa, Iowa, USA, in 1996 under the guidance of Professor John P. N. Rosazza. After his postdoctoral research with Professor Edward A. Dennis at University of California, San Diego, he worked as a Senior Scientist at MicroGenomics, Inc., California, USA from 1998 to 1999. From 1999 to 2006, he served as a Senior Research Investigator at Lead Discovery and Enzyme Technology Departments of Bristol-Myers Squibb Pharmaceutical Research Institute in New Jersey, USA. Since 2007, he has been appointed as an Endowed Professor and Director of Laboratory of Chemical Biology at China Pharmaceutical University, and he presently is also an Adjunct Professor of Department of Chemical Biology, Rutgers University, USA. Currently, he has published over 80 peer-reviewed papers and invented more than 40 patents. The research of Dr. Chen’s group focuses on discovery and validation of novel antitumor targets using chemical proteomics approach, elucidation and manipulation of biosynthetic pathways for microbial secondary metabolites and development of biocatalytic routes for chiral drug intermediates. 

Journal Reference

Anal Biochem. 2016 Jun 1;502:16-23.

Wu X1, Zhu S1, Huang P1, Chen Y2.

Show Affiliations
  1. State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, 210009, People’s Republic of China.
  2. State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, 210009, People’s Republic of China. Electronic address: [email protected]

Abstract

MicroRNA (miRNA) plays vital roles in various biological processes. In general, sensitivity and specificity are the major parameters for the quantification of miRNA. In this study, padlock probe-rolling circle amplification and Förster resonance energy transfer (pRCA-FRET) were coupled for specific and quantitative detection of miRNA. pRCA-FRET showed superior specificity to differentiate single-base mismatch and excellent sensitivity with a detection limit of 103 aM. The current method has the potential to quantify low amounts of miRNA in the same family for studies on their biological functions.

Copyright © 2016 Elsevier Inc. All rights reserved.

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