4.8 Article

Isothermal digital detection of microRNAs using background-free molecular circuit

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SCIENCE ADVANCES
卷 6, 期 4, 页码 -

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AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/sciadv.aay5952

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资金

  1. Universite de Recherche Paris Sciences et Lettres (PSL)
  2. ESPCI-Paris
  3. Universite Paris-Descartes
  4. Centre National de la Recherche Scientifique (CNRS)
  5. Institut National de la Sante et de la Recherche Medicale (INSERM)
  6. Ligue Nationale Contre le Cancer (LNCC, Program Equipe labelisee LIGUE) [EL2016.LNCC/VaT]
  7. European Research Council [647275 ProFF, 780519 DeepMiR]
  8. ITMO Cancer within the Frontiers in Life Science PhD program (FdV)
  9. SIRIC CARPEM
  10. Physicancer program [PC201423]

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MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.

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