Isothermal digital detection of microRNA using background-free molecular circuit

Abstract

MicroRNA, 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 method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design DNA circuit that converts, threshold, amplifies and report the presence of a specific microRNA, down to the femtomolar concentration. Using a leak-absorption mechanism, we were able to suppress non-specific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNA: 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.

Multiplex Digital MicroRNA Detection Using Cross-Inhibitory DNA Circuits

“…To detect minute quantities of these short sequences, we have reported a nickase−polymerase−exonuclease system whose sensitivity has been boosted (limit of detection < 10 fM), thanks to a leakabsorption mechanism, which counterbalances nonspecific amplification caused by primer-independent polymerization. 28 In this system, a typical singleplex amplification mixture is composed of four DNA templates, each designed to perform a specific function: a conversion template (cT, e.g., mir39toα), which, upon hybridization to its cognate microRNA target (e.g., mir39), linearly produces a 12-mer oligonucleotide trigger (e.g., α) using polymerization/nicking cycles. The trigger, in turn, stimulates a bistable amplification switch made of an amplification template (aT, e.g., aTα) and a pseudotemplate (pT, e.g., pTα).…”

“…For example, a bistable circuit can be used to install ultrasensitivity in DNAbased signal amplification schemes. 28 Here, our approach is built on a tetrastable circuit that stabilizes the two asymmetric ON states necessary for duplex detection and avoids nonspecific response from one target to the other. Our design uses DNA-encoded cross-inhibitors, solving the challenge of balancing the by having the strength of inhibition reactions controlled by strand concentrations.…”

“…In the present study, we start from a solution-phase isothermal approach to detect microRNA, which is based on signal amplification using reprogrammable molecular circuits. This procedure has recently been adapted in a digital format, 28 and we wished to explore its multiplexing potential. We first show that molecular cross-talks generate unwanted interferences between multiplexed detection channels.…”

Streamlined digital bioassays with a 3D printed sample changer

“…To demonstrate the usefulness of our 3D printed device on practical applications, we performed the digital detection of 11 microRNA targets from a human colon extract, using a previously reported isothermal and digital quantification method ( Figure 4a) 54 . Briefly, 4 oligonucleotides (a converter template, an amplification template, a pseudotemplate and a reporting template) and 5 enzymes (a DNA polymerase Vent(exo-), two nicking enzymes Nt.BstNBI and Nb.BsmI, an exonuclease ttRecJ and a restriction enzyme BsmI) are required to exponentially amplify a fluorescent signal from single microRNAs isolated in microdroplets.…”

“…Based on a 3D printed sample tray, the sample changer allows for the serial emulsification of up to 15 samples using a single chip, with significantly improved time and reagent usage. This is validated through the simultaneous digital quantification of multiple microRNAs from cell extracts using a recently reported leak-free isothermal amplification chemistry 54 . We discuss potential carry-over contamination issues and shows that they can be efficiently mitigated by introducing Figure 1.…”