Multiplex Digital MicroRNA Detection Using Cross-Inhibitory DNA Circuits

Abstract

Ubiquitous post-transcriptional regulators in eukaryotes, microRNAs are currently emerging as promising biomarkers of physiological and pathological processes. Multiplex and digital detection of microRNAs represents a major challenge toward the use of microRNA signatures in clinical settings. The classical reverse transcription polymerase chain reaction quantification approach has important limitations because of the need for thermocycling and a reverse transcription step. Simpler, isothermal alternatives have been proposed, yet none could be adapted in both a digital and multiplex format. This is either because of a lack of sensitivity that forbids single molecule detection or molecular cross-talk reactions that are responsible for nonspecific amplification. Building on an ultrasensitive isothermal amplification mechanism, we present a strategy to suppress cross-talk reactions, allowing for robust isothermal and multiplex detection of microRNA targets. Our approach relies on target-specific DNA circuits interconnected with DNA-encoded inhibitors that repress nonspecific signal amplification. We demonstrate the one-step, isothermal, digital, and simultaneous quantification of various pairs of important microRNA targets.

A small-molecule chemical interface for molecular programs

“…Here we decided to replace the B11 output of the psT with the complementary sequence antiB11 (aB11). For clarity we named these repressing input modules protein sensing killer Template (pskT), in line with recently described killer Templates (kT) ( 25 ) (Figure 2C , E ). At the same time, the mixture was complemented with an unconditional mechanism to produce the signal B11, which we call a source template (sT).…”

“…We started with the PEN-DNA framework ( 22 ), a powerful molecular programming language which has allowed the rational building of a variety of molecular systems, including batch oscillators ( 23 ), programmable pattern formation ( 24 ), or ultrasensitive multi-input sensors ( 25 ). The PEN-DNA employs Polymerase, Exonuclease and Nickase (PEN) enzymes as the chemical machinery.…”

“…Above a certain concentration threshold, this sink gets saturated and exponential amplification ensues. The combination aT/pT thus creates a bistable switch that can be adapted to sensitive detection of a variety of inputs ( 25 , 28 , 29 ).…”

“…Similar switches have already been used for the sensitive detection of nucleic acids, down to the femtomolar range ( 25 , 28 ). However, these circuits were typically run at temperature ranging from 45 to 50°C, which is not appropriate if one wishes to use them to detect, or in combination with, protein components coming from a mesophilic organism.…”

Interrelationship between miRNA and splicing factors in pancreatic ductal adenocarcinoma

“…Since multiplexing capability plays a crucial role in miRNAs detection, in addition to the abovementioned multiplexing approaches, it is worth citing the suspension arrays (i.e., on-particle), which represent promising emerging methods for highly multiplex analysis of complex samples due to the versatility of the encoded microspheres used in conjunction with flow cytometry [133]. Furthermore, Rondelez et al recently reported an isothermal amplification mechanism for multiplex and digital detection of miRNAs using the rational building of a molecular circuit that suppresses non-specific amplification due to cross-talk reactions [134]. In conclusion, extensive efforts have been made so far to develop efficient and sensitive methods for miRNA detection, but there still remains a need for a standardized method that should be highly sensitive, specific and multiplexable.…”