RCA-enhanced multifunctional molecule beacon-based strand-displacement amplification for sensitive microRNA detection

Abstract

MicroRNAs (miRNAs) are regarded as promising cancer-related biomarkers. Here, we developed a simple, sensitive and specific fluorescence method based on the combination of rolling circle amplification (RCA) and multifunctional molecular beacon-based strand-displacement amplification (MMB-SDA) for the amplification detection of let-7a miRNA. Specifically, a multifunctional molecular beacon (MMB) was proposed to execute several functions, including the signal reporter, primer and polymerization template. To achieve RCA reaction, 3′ and 5′ terminal bases of padlock probe were designed as the recognition regions capable of hybridizing with let-7a miRNA. Moreover, this miRNA could serve as the primer to trigger RCA reaction after ligation, producing tens and hundreds of tandemly repeated copies of cycle. Subsequently, the RCA product hybridized with MMB and initiated the repetitive SDA reaction, opening a significant amount of MMBs and causing an amplified signal. As a result, let-7a miRNA can be specifically detected down to 51 pM, and the single-base difference between let-7a miRNAs was easily detected. The potential application was demonstrated via evaluating the let-7a miRNA level in real samples (total RNA extracted from HeLa cells). Therefore, the developed sensing strategy would provide a powerful platform for early clinical diagnostics.