The fabrication of 1,2-dicarbonyl compound-caging isothermal exponential amplification and its application in highly sensitive detection of tumor exosomal miRNA

Abstract

The exponential amplification reaction (EXPAR) possesses the advantage of high amplification efficiency of producing large amounts of short nucleic acids in a short time. However, primer-independent DNA autosynthesis and ab initio DNA self-synthesis mediated nonspecific amplification increase the risk of high background signal, which limits its application in the fabrication of ultrasensitive sensors. Here, we developed a new strategy called 1,2-dicarbonyl compound-caging EXPAR (caging-EXPAR) by innovatively modifying EXPAR templates with 1,2-dicarbonyl compounds. Five 1,2-dicarbonyl compounds including glyoxal and ninhydrin were chosen, all of which were proved to specifically bind to guanosine on EXPAR template, efficiently reducing the background amplification of EXPAR and exhibiting an obviously improved discrimination between target and background. The possible mechanisms of the role of 1,2-dicarbonyl compounds played in EXPAR were proposed, and three possible factors which might induce the serious nonspecific amplification were investigated and verified. Caging-EXPAR provided a general, simple, convenient and beneficial strategy to slow down the generation of EXPAR background signals. Based on this method, choosing miRNA let-7a as a model, the minimum detectable amount was 10 zmol, which was reduced by 3 orders of magnitude compared to traditional EXPAR. More importantly, the strategy was successfully applied to monitor the expression level of the low-abundant miRNA in MCF-7 cell derived exosomes. This highly portable and cost-effective method can not only promote the real-time quantitative detection of specific nucleic acids in many fields such as clinical diagnosis and prognostic treatment, but also provide a complementary theoretical support for EXPAR background amplification, which is conducive to the improvement and wide application of other amplification reactions.