One-Pot Detection of miRNA by Dual Rolling Circle Amplification at Ambient Temperature with High Specificity and Sensitivity

Rolling circle amplification (RCA) at ambient temperature is prone to false positive signals during nucleic acid detection, which makes it challenging to establish an efficient RCA detection method. The false positive signals are primarily caused by binding of non-target nucleic acids to the circular single-stranded template, leading to non-specific amplification. Here, we present an RCA method for miRNA detection at 37 °C using two circular ssDNAs, each of which is formed by ligating the intramolecularly formed nick (without any splint) in a secondary structure. The specific target recognition is realized by utilizing low concentrations (0.1 nM) of circular ssDNA1 (C1). A phosphorothioate modification is present at G*AATTC on C1 to generate a nick for primer extension during the primer self-generated rolling circle amplification (PG-RCA). The fragmented amplification products are used as primers for the following RCA that serves as signal amplification using circular ssDNA2 (C2). Notably, the absence of splints and the low concentration of C1 significantly inhibits non-target binding, thus minimizing false positive signals. A high concentration (10 nM) of C2 is used to carry out linear rolling circle amplification (LRCA), which is highly specific. This strategy demonstrates a good linear response to 0.01-100 pM of miRNA with a detection limit of 7.76 fM (miR-155). Moreover, it can distinguish single-nucleotide mismatch in the target miRNA, enabling the rapid one-pot detection of miRNA at 37 °C. Accordingly, this method performs with high specificity and sensitivity. This approach is suitable for clinical serum sample analysis and offers a strategy for developing specific biosensors and diagnostic tools.