Ultrasensitive and Multiplex Detection of RNAs with Single-Nucleotide Resolution Using Lambda Exonuclease-Assisted Probe Signal Amplification-Rolling Circle Amplification

Rolling circle amplification (RCA) is a simple and efficient isothermal enzymatic reaction. However, its linear amplification kinetics and unsatisfactory multiplex detection capability severely limit further improvements in detection sensitivity and application flexibility. In this work, we developed a lambda exonuclease-assisted probe signal amplification strategy termed LEPA, integrated with endonuclease-mediated exponential RCA for the sensitive detection of target nucleic acids. This system enables precise target recognition through ligase and padlock probes, which, in turn, initiate cascaded RCA for efficient signal amplification. Through a cyclic “binding-cleavage-dissociation” process between E-probes and amplification products, the system achieves dual signal amplification and single-nucleotide resolution. The LEPA-RCA system demonstrates exceptional detection sensitivity and specificity, allowing precise discrimination of alleles with a mutation frequency as low as 0.1% and supporting multiplex intracellular RNA detection. In clinical validation, this method effectively distinguished the expression levels of serum miR-16-5p and miR-92a-3p between healthy individuals and colorectal cancer (CRC) patients, showing high consistency with RT-qPCR results. Moreover, it achieved excellent diagnostic performance (AUC = 0.85). With a modular design, the LEPA-RCA system serves as a robust platform for high-precision multiplex nucleic acid detection in complex biological matrices, showing great potential for clinical translation.