title: Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zinc Finger Protein Specific to DNA-RNA Hybrids.
abstract: Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just Another Zinc finger proteins (JAZ, ZNF346) bind preferably (but nonsequence-specifically) to DNA-RNA hybrids over single-stranded RNAs, single-stranded DNAs, and double-stranded DNAs. We present an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA-RNA hybrid formed between a DNA capture probe and a target miRNA-21. This enables us to use chemically stable DNA as a capture probe instead of RNA as well as to apply a standard sandwich-type assay format to miRNA detection. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical-chemical-chemical (ECC) redox cycling involving an ALP product (hydroquinone). Low nonspecific adsorption of ALP-conjugated JAZ is obtained using a polymeric self-assembled-monolayer-modified and casein-treated indium-tin oxide electrode. The detection method can discriminate between target miRNA-21 and nontarget nucleic acids (DNA-DNA hybrid, single-stranded DNA, miRNA-125b, miRNA-155, single-base mismatched miRNA, and three-base mismatched miRNA). The detection limits for miRNA-21 in buffer and 10-fold diluted serum are approximately 2 and 30 fM, respectively, indicating that the detection method is ultrasensitive. This detection method can be readily extended to multiplex detection of miRNAs with only one ALP-conjugated JAZ probe due to its nonsequence-specific binding character. We also believe that the method could offer a promising solution for point-of-care testing of miRNAs in body fluids.
authors.affiliation: Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University , Busan 46241, Korea.
authors.forename: Chiew San
authors.initials: CS
authors.lastname: Fang
authors.affiliation: Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University , Busan 46241, Korea.
authors.forename: Kwang-Sun
authors.initials: KS
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST) , Daejeon 34141, Korea.
authors.forename: Byeongjun
authors.initials: B
authors.lastname: Yu
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST) , Daejeon 34141, Korea.
authors.forename: Sangyong
authors.initials: S
authors.lastname: Jon
authors.affiliation: Department of Chemistry, Western Kentucky University , Bowling Green, Kentucky 42101, United States.
authors.forename: Moon-Soo
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University , Busan 46241, Korea.
authors.forename: Haesik
authors.initials: H
authors.lastname: Yang
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D055664:Electrochemical Techniques / Q000379:methods* / Q000592:standards; D004566:Electrodes; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis*; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D016601:RNA-Binding Proteins; C121935:ZNF346 protein, human; D012313:RNA; D004247:DNA; D000469:Alkaline Phosphatase