One-Pot and Enzyme-Free Analysis of mRNA Vaccines Based on a Double Amplification System Featured by Adjustable Structure MNAzyme

mRNA vaccines have been developed as a captivating and highly prospective avenue for the advancement of immunotherapy. With the advancement of antitumor mRNA vaccines into clinical trials, it is important to monitor the levels of nucleic acid drugs. In this study, we have engineered a new biosensor for melanoma-associated antigen mRNA vaccines utilizing an adjustable structure MNAzyme (ASM) activated by an entropy-driven circuit (EDC). The EDC process is specifically triggered by the target mRNA through the exposed toehold, achieving initial amplification. As a result of proximity effects facilitated by the EDC products, ASM is successfully assembled and activated by undergoing conformational transformation because of rational sequence design, continuously cleaving the fluorescence reporter and realizing the second round of signal amplification. Compared with conventional DNAzyme, ASM can effectively reduce signal leakage and concurrently preserve cleavage efficiency, benefiting from the stable presence of a stem-loop hairpin and an adjustable molecular switch. Moreover, composed only of DNA with highly integrated sequences, this method could realize detection in an enzyme-free, one-step, and one-pot detection manner. Finally, EDC-ASM demonstrates high programmability, excellent specificity, and sensitivity in nucleic acid detection, providing a novel strategy for the study of antitumor mRNA vaccines.