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  • br Murtola T J Syv l H

    2020-07-27


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    130 MOLECULAR METABOLISM 25 (2019) 119e1302019 University Health Network. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
    www.molecularmetabolism.com
    Contents lists available at ScienceDirect
    Analytica Chimica Acta
    An amplification strategy using DNA-Peptide dendrimer probe and mass spectrometry for sensitive MicroRNA detection in breast cancer
    a School of Pharmacy, Nanjing Medical University, Nanjing, 211166, China
    b China State Key Laboratory of Reproductive Medicine, Nanjing, 210029, China
    c Department of Pharmacy, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China
    d School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China
    A signal amplification strategy for sensitive miRNA detection was developed.
    DNA-peptide dendrimer probe con-tains eight reporter peptides. MiRNA signal was converted to mass response of reporter peptide via the probe.
    The assay provides the possibility for quantitative analysis of miRNA.
    Article history:
    Received in revised form
    Keywords:
    MiRNA
    Sensitive quantification DNA-Peptide dendrimer (DPD) probe Liquid chromatography-tandem mass spectrometry (LC-MS/MS) Signal amplification
    Breast cancer
    MicroRNAs (miRNAs) are emerging as novel biomarkers for diagnosis and treatment of various cancers, including breast cancer. Because the value of biomarkers primarily depends on whether they are quantifiable, great effort has been taken to develop assays for sensitive and accurate quantification of miRNAs. However, most of current assays have high nonspecific amplification effect, which limits quantification accuracy. In this study, we circumvented copying of nucleic acid sequence and developed a signal amplification strategy based on a novel DNA-peptide dendrimer (DPD) probe coupled with mass spectrometry. The DPD probe RP8-MAP4-DNA contained three functional domains, including substrate peptides containing eight reporter peptides and tryptic cleavage sites, peptide dendrimer scaffold and DNA complementary to target miRNA. The probe was first hybridized with the target miRNA (i.e., miR-
    21) that was biotinylated and attached to streptavidin agarose in advance. After trypsin digestion, the reporter peptide was liberated and quantified using LC-MS/MS. The signal intensity was approximately 8 fold greater than that without signal amplification. Finally, the developed assay was applied for the quantitative analysis of miR-21 in 3 human breast cell lines and 102 matched pairs of breast tissue samples. The miR-21 expression in tissue was also evaluated depend on histopathological features, molecular subtypes and prognosis of breast cancer. The result demonstrated that combination of DPD probe and mass spectrometry is a promising strategy for quantification of miRNAs and illustration of their biomarker potential, especially those at low abundance.
    * Corresponding author. School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing, 211166, China. E-mail address: [email protected] (Y. Chen).