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 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.

Spatiotemporal Patterning of Living Cells with Extracellular DNA Programs

“…Ultimately, reactive media could be integrated into smart bandages 8 or into engineered living materials 58 but also function as a detector, 57 as an actuator, or both, while benefiting of the computation capabilities of DNA.…”

A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics

“…Artificial nucleic acid circuits have attracted much attention in the fields of bioanalysis, bioregulation, and bioengineering due to the programmability and scalability of Watson-Crick base pairing and the diverse enzymatic or functional nucleic acid toolkits (1,2,5).…”

“…Among these circuits, the catalytic nucleic acid circuits integrate cascaded or cycling reactions to generate multiple outputs in response to a single input, providing valuable toolkits for signal amplification (2). Using nucleic acid-based molecular programming, various catalytic circuits, such as polymerase/exonuclease/nickase DNA circuit (5), self/crosscatalytic nucleic acids (6,7), hybridization chain reaction (8), and catalytic hairpin assembly (3), have been developed. In addition, their dynamics are powered by DNA-manipulating enzymes, DNAzymes/ RNAzymes, or strand displacement reactions.…”

Directed Evolution of Enzymes based on in vitro Programmable Self-Replication

“…We selected NBI as a target because of its many applications, including molecular diagnostics 30,31 and molecular technologies 2,33 . NBI is currently used in its natural form, although many endonucleases have been engineered for better performance in vitro 34 .…”