Protease-Hydrolysis-Driven Approach towards the Quantification of Cellular mRNA after Drug Treatment in Protein Nanopores

Abstract

Recently, the exploration of potential therapeutic applications for approved pharmaceuticals arouses significant interest. Messenger RNA (mRNA), which intricately involves in biological processes and reflects the physiological states of organisms, serves as a robust biomarker for investigating the impacts of drugs on cellular environments in both cell biology and clinical research. Nanopore technology has demonstrated to be a versatile tool for multiple applications, including disease diagnostics, environmental monitoring, and food surveillance. Herein, we proposed a protease-hydrolysis-based approach for mRNA quantification within protein nanopores. In this context, a type of peptide-DNA chimera probe was synthesized through a click reaction. The presence of target mRNA molecules promoted the formation of a sandwich complex between the target and probes, which in turn enabled the protease-mediated cleavage of the peptide segment. Then, the cleaved peptide fragments served as external probes for nanopore analysis. To optimize the measurement conditions, the effects of voltage bias and buffer pH values on the performance of the nanopore sensor were systematically investigated. The proposed nanopore sensor was further performed to assess cellular samples, which showed a ∼4-fold reduction in P2RX7 mRNA levels in the SMMC-7721 cell line pre- and post-carrimycin therapy. This outcome indicated the anti-cancer efficacy of carrimycin, and the result was comparable to that of qRT-PCR analysis, highlighting its reliable performance in real sample detection. This protein nanopore mRNA sensor successfully addressed high molecular weight and complex background species in the quantification of mRNA. Our study introduced an innovative methodology for cellular mRNA quantification to explore the potential therapeutic application of pharmaceuticals, while also offering a new approach for mRNA quantification in disease diagnosis, toxicological assessment, and personalized medicine.