Base gap switches as transcriptional regulators and their applications as biosensors

Transcriptional and translational regulators ('switches') function as biosensors by controlling how gene circuits respond to specific stimuli. However, contemporary switches suffer from spontaneous activation or off-target responses, limiting sensitivity and specificity. Here we introduce base gap switches, a class of programmable transcriptional regulators that are selectively activated by DNA. Base gap switches are simple to design and install, provide tight control of gene expression and exhibit high specificity and orthogonality. They can discriminate single-nucleotide mismatches and integrate with nucleic acid amplification methods. Using base gap switches, we developed an assay for Trichomonas vaginalis with a limit of detection of 0.6 copies μl^-1 and validated its performance using clinical samples. We further demonstrate a one-pot assay for differentiating Neisseria gonorrhoeae and Chlamydia trachomatis, and an assay for detecting single-nucleotide variants of EGFR-associated cell-free DNA. These results establish base gap switches as a versatile tool for gene circuit-based biosensing.