Characterization of artificial riboswitches for Coxsackievirus B3 detection using machine learning

With the increase of recycled water to offset water demand, the potential possibility to spread contagious RNA viruses, such as Coxsackievirus B3, increases. However, detection of viral particles remains challenging because of low viral concentrations in wastewater and high mutation rates of the RNA virus. Robust monitoring is needed with low cost and low infrastructure technologies to increase accessibility of monitoring technologies worldwide. Novel viral detection methods have been developed, based on synthetic riboswitches that bind to the target virus and trigger a reporter gene, thus amplifying the detection signals. Such monitoring technologies can leverage machine learning to optimize the candidate nucleic acid sequences used for detection. To support the design of effective riboswitches, we present a machine learning model for classifying riboswitch performance, integrating RNA sequence data with secondary structural features based on free energy calculations and parameters that represent single strandedness. This model uses a sparsely gated Mixture of Experts (MoE) architecture to route sequence and thermodynamic features to specialized experts, achieving strong generalization performance across cross-validation and held-out testing. When evaluated against baseline Decision Tree, Random Forest, Gaussian Naive Bayes, and Dense Neural Network classifiers, the MoE model demonstrated near-zero classification errors. Furthermore, post-hoc feature importance and k-mer analyzes reveal that near-perfect predictive performance in silico is strongly driven by sequence constructs in addition to generalizable folding mechanics. An ablation study of numerical feature and sequence inputs showed that while the Dense Neural Network also achieved high accuracy across all ablation conditions, the MoE architecture was retained as the primary model because its specialized subnetworks do not require all model parameters to be activated offering a potential computational advantage.