De novo-designed ribozyme-controlled riboregulator for cell-free diagnostics

Abstract Cell-free systems have great potential for nucleic acid assays, yet universal onsite diagnostics without preamplification are limited. Here, a universal cell-free diagnostic platform termed TRACKer (Target-Responsive non-preAmplification Cell-free diagnostic Kit) has been developed for detecting multiple respiratory viral RNAs in laboratory and field settings. TRACKer integrates three modules: ribozyme allostery module, riboregulator activation module, and output module. The ribozyme allostery module enables universal target recognition through strand displacement-mediated ribozyme conformational switching. The riboregulator activation module achieves preamplification-free detection via cascade expression of reporter proteins. The output module provides swappable reporter templates for luminescent quantification and lateral flow visualization in diverse scenarios. TRACKer is a robust system that enables rapid ( < 70 min) nucleic acid detection without preamplification and demonstrates attomolar sensitivity (1-10 aM) for six respiratory viruses. Overall, TRACKer presents a promising approach to nucleic acid detection, offering a low-cost and scalable solution with potential applications in point-of-care diagnostics and beyond.