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Isothermal digital detection of microRNAs using background-free molecular circuit

Abstract : MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising diseasespecific 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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https://hal.archives-ouvertes.fr/hal-03058004
Contributor : Yannick Rondelez <>
Submitted on : Monday, January 4, 2021 - 10:25:40 AM
Last modification on : Thursday, June 3, 2021 - 10:44:43 AM
Long-term archiving on: : Monday, April 5, 2021 - 7:08:55 PM

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Guillaume Gines, Roberta Menezes, Kaori Nara, Anne-Sophie Kirstetter, Valérie Taly, et al.. Isothermal digital detection of microRNAs using background-free molecular circuit. Science Advances , American Association for the Advancement of Science (AAAS), 2020, 6 (4), pp.eaay5952. ⟨10.1126/sciadv.aay5952⟩. ⟨hal-03058004⟩

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