Isothermal Amplification on a Structure-Switchable Symmetric Toehold Dumbbell-Template: A Strategy Enabling MicroRNA Analysis at the Single-Cell Level with Ultrahigh Specificity and Accuracy

Accurate analysis of microRNAs (miRNAs) at the single-cell level seriously requires analytical methods possessing extremely high sensitivity, specificity and precision. By rational engineering of a structure-switchable symmetric toehold dumbbell-template (STD-template), we propose a novel isothermal symmetric exponential amplification reaction (SEXPAR) method. The sealed and symmetric structure of the STD-template allows exponential amplification reaction (EXPAR) to occur upon every annealing of target miRNA without loss of amplification efficiency. In addition, the rigid and compact structure of the STD-template with an appropriate standard free energy ensures SEXPAR only be activated by target miRNA. As a result, the SEXPAR method isothermally quantified let-7a down to 0.01 zmol (6.02 copies per 10 mu L) with an ultrahigh specificity which is efficient enough to discriminate one-base-mismatched miRNAs, and a remarkably high precision even for the determination of 6.02 copies let-7a (the standard deviation was reduced from >60% down to 23%). The dynamic range was also extended to 10 orders of magnitude. The method was successfully applied for the determination of let-7a in human tissues, sera and even single-cell lysate, with obviously better precision than quantitative reverse transcription polymerase chain reaction (RT-qPCR) and other EXPAR-based methods. The SEXPAR method may serve as a powerful technique for the biological research and biomedical studies of miRNAs and other short nucleic acids.