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.

中文翻译：

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使用交叉抑制性DNA电路进行多重数字MicroRNA检测。

真核生物中无处不在的转录后调节剂，微RNA目前正在成为生理和病理过程的有前途的生物标志物。microRNA的多重和数字检测代表了在临床环境中使用microRNA签名的主要挑战。由于需要热循环和逆转录步骤，因此经典的逆转录聚合酶链反应定量方法具有重要的局限性。已经提出了更简单的等温替代方案，但是没有一种可以同时采用数字和多路复用格式。这是由于缺乏灵敏度而导致无法进行单分子检测的原因，或是由于造成非特异性扩增的分子串扰反应。建立在超灵敏的等温放大机制上，我们提出了一种抑制串扰反应的策略，允许对microRNA目标进行强大的等温和多重检测。我们的方法依靠与DNA编码的抑制剂互连的靶标特异性DNA电路，该抑制剂抑制非特异性信号放大。我们展示了重要的microRNA靶的各对的一步，等温，数字和同时定量。