z Analytical Chemistry Photoinduced Electron Transfer-Based Fluorescence Quenching Combined with Rolling Circle Amplification for Sensitive Detection of MicroRNA Fulin Zhou, Rong Meng, Qiang Liu, Yan Jin, and Baoxin Li* [a] MicroRNAs (miRNAs) detection is crucial for further under- standing the biological functions of miRNAs and early cancer diagnosis and therapeutics, but now remains a great challenge. Here, we present a novel homogenous biosensing strategy for fluorescence detection of miRNA. In this strategy, a preliminary synthesized circular DNA was used as recognition probe for hybridization with miRNA target, and then miRNA target primed a rolling circle amplification (RCA) reaction. The RCA product could be hybridized with thousands of carboxyfluor- escein (FAM)-labeled linear DNA probes, which led FAM to be close to -GGG- base of RCA product, accompanying with the significant fluorescence quenching due to photoinduced elec- tron transfer (PET) between FAM and guanine. This is for the first time to integrate RCA and PET into one detection process. With highly efficient amplification of RCA and excellent signal readout of PET, this method exhibited a high sensitivity toward target miRNA with a detection limit of 6 aM. The target- dependent circularization of the padlock probe and the ligation reaction could improve the specificity effectively, leading to discrimination between miRNA family members. This method provides a simple, isothermal, and low-cost approach for sensitive detection of miRNA and holds great potential for early diagnosis in gene-related diseases. Introduction MicroRNAs (miRNAs) are a class of endogenous, non-protein coding small RNA molecules (19–25 nucleotides), which act as post-transcriptional regulators of gene expression and play an important role in many biological processes. [1] Recent studies have found that some miRNAs have altered expression in cancer cells; abnormal expression of miRNAs has been con- firmed to be highly related to the development of most types of cancers. [2, 3] Nowadays, miRNAs have been regarded as biomarkers and therapeutic targets in cancer treatment. [4, 5] So, accurate and quantitative detection of miRNAs is crucial for further understanding the biological functions of miRNAs and early cancer diagnosis and therapeutics. [6] However, it is difficult to analyze the miRNAs because of their unique characteristics, including their small size, sequence homology among family members, and low abundance in total RNA samples. [7] So, the strategies for specific and sensitive quantitative detection of miRNAs are in urgent need. [8-11] Quantitative reverse-transcription polymerase chain reac- tion (qRT-PCR) is the gold standard for miRNA quantifica- tion. [12, 13] The qRT-PCR technique offers good sensitivity and specificity for miRNA detection. But, qRT-PCR generally requires the multiple primer design and the precise temperature control, which increases the experimental cost and complexity. In recent years, various isothermal amplification techniques, which can be performed at one reaction temperature under simple experimental conditions (e. g. in water bath), have been developed as alternatives to PCR. [14-17] Among these isothermal amplification techniques, rolling circle amplification (RCA) has become increasing popular for the quantitative detection of miRNA. [7, 18-27] In RCA process, a short DNA (or RNA) is amplified to form a long single-stranded DNA (ssDNA) in the presence of a circular DNA template and special DNA polymerase. The RCA product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template. A single molecule can be amplified hundreds to thousands of times of template as a fundamental characteristic of RCA. So, the target molecule can be ultra-sensitively detected by detecting RCA product. The power, simplicity, specificity and versatility of RCA technique have made it an attractive tool for biomedical research. [28] For miRNA detection, miRNAs are too short to be used as PCR templates, but ~ 20 nt in length is able to bind RCA substrates to initiate the RCA processes. Therefore, target miRNA can be directly used as a primer to initiate the tandem repeated DNA product of RCA. Then, the detection of miRNA is converted to the assay of the tandem-repeated DNA that can be achieved by various signal readout techniques, such as fluorescence, [18-20] chemilumines- cence, [21] electrochemiluminescence [25, 26] and colorimetry. [29] It is very desirable to quantify the RCA products in a real-time homogeneous fashion. As one very popular fluorescence probe for homogeneous assay, molecular beacon (MB) has garnered much attention in the RCA-based biosensing platform. [30-37] MB [a] F. Zhou, R. Meng, Q. Liu, Y. Jin, B. Li Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Pro- vince School of Chemistry & Chemical Engineering Shaanxi Normal University, Xi’an 710062, China Fax: + 86-29-81530727 E-mail: libaoxin@snnu.edu.cn Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/slct.201601485 Full Papers DOI: 10.1002/slct.201601485 6422 ChemistrySelect 2016, 1, 6422 – 6428  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim