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The structure and mechanism of the bacterial enzyme β-lactamase have been well-studied due to its clinical role in antibiotic resistance. β-Lactamase is known to hydrolyze the β-lactam ring of the cephalosporin scaffold, allowing a spontaneous self-immolation to occur. Previously, cephalosporin-based sensors have been developed to evaluate β-lactamase expression in both mammalian cells and zebrafish embryos. Here, we present a circular caged morpholino oligonucleotide (cMO) activated by β-lactamase-mediated cleavage of a cephalosporin motif capable of silencing the expression of T-box transcription factor Ta (tbxta), also referred to as no tail a (ntla), eliciting a distinct, observable phenotype. We explore the use of β-lactamase to elicit a biological response in aquatic embryos for the first time and expand the utility of cephalosporin as a cleavable linker beyond targeting antibiotic-resistant bacteria. The addition of β-lactamase to the current suite of enzymatic triggers presents unique opportunities for robust, orthogonal control over endogenous gene expression in a spatially resolved manner.

MicroRNAs (miRNAs) are noncoding RNA molecules of 22−24 nucleotides that are estimated to regulate thousands of genes in humans, and their dysregulation has been implicated in many diseases. is the most abundant miRNA in the liver and has been linked to the development of hepatocellular carcinoma and hepatitis C virus (HCV) infection. Its role in these diseases renders miR-122 a potential target for small-molecule therapeutics. Here, we report the discovery of a new sulfonamide class of small-molecule miR-122 inhibitors from a high-throughput screen using a luciferase-based reporter assay. Structure−activity relationship (SAR) studies and secondary assays led to the development of potent and selective miR-122 inhibitors. Preliminary mechanism-of-action studies suggest a role in the promoter-specific transcriptional inhibition of miR-122 expression through direct binding to the liver-enriched transcription factor hepatocyte nuclear factor 4α. Importantly, the developed inhibitors significantly reduce HCV replication in human liver cells.

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