Palindromic hyperbranched rolling circle amplification enabling ultrasensitive microRNA detection

We herein describe a palindromic hyperbranched rolling circle amplification (PH-RCA) reaction and its application for ultrasensitive detection of microRNAs (miRNAs). In this strategy, target miRNAs bind to a dumb-bell probe (DP) and initiate the RCA reactions, concomitantly converting the dumb-bell structure to the circular form, which then allows the annealing of the palindromic primers to promote an additional two RCA reactions. As a consequence of the RCA reactions promoted by both target miRNAs and palindromic primers, multiple long concatenated DNA strands would be produced. Importantly, the palindromic primers can also bind to numerous palindromic domains of the long linear single DNA strands, consequently promoting highly branched simultaneous extension reactions at multiple sites. By detecting the fluorescence signals resulting from the amplified DNA products, we successfully identified target miRNA under isothermal conditions with excellent specificity. The PH-RCA technique developed in this work would greatly advance the conventional RCA reaction and HRCA reaction by significantly enhancing the sensitivity and reducing the reaction time within 30 min.