In cellulo monitoring of mRNA expression in Euglena gracilis using spherical nucleic acid (SNA) nanoflare

Abstract

Detection of metabolic products, precursors, and gene expression in living cells is crucial for advancing industrial microbial applications and understanding in cellulo biological processes. In this study, a gold nanoparticle-based spherical nucleic acid (SNA) nanoflare was employed to detect mRNA in Euglena gracilis cells. A SNA nanoflare targeting EgGSL2, a key gene in paramylon biosynthesis, was designed using a 30-base oligonucleotide specific to EgGSL2 mRNA. Fabrication of SNA nanoflare was confirmed through dynamic light scattering (DLS) and UV–visible spectroscopy. In vitro validation revealed an up to 32-fold increase in fluorescence in the presence of the target mRNA, demonstrating high specificity and sensitivity, while scramble controls exhibited negligible signals. In cellulo detection of EgGSL2 mRNA in E. gracilis was then performed by comparing wild-type and EgGSL2 knockdown cells, confirming effective discrimination of expression states of EgGSL2 mRNA, which was consistent with qPCR results. Receiver operating characteristic (ROC) curve analysis further demonstrated an AUC of 0.92 in binary classification based on fluorescence imaging data. Following 12 and 24 h of SNA nanoflare exposure, a moderate increase in reactive oxygen species (ROS) was observed, while paramylon production remained unchanged, indicating moderate but tolerable cellular stress. These findings highlight the potential of SNA nanoflares as a sensitive, non-invasive molecular sensing tool for the discrimination of gene expression states in living cells, with promising applications in cellular diagnostics and bioengineering.