Covalent aptamers are novel biochemical tools for fast and selective transfer of labels to target proteins. Equipped with cleavable electrophiles, these nucleic acid probes enable the installation of functional handles onto native proteins. The high affinity and specificity with which aptamers bind their selected targets allows for quick, covalent labeling that can compete with nuclease-mediated degradation. Here, we introduce the first application of covalent aptamers to modify a specific cell surface protein through proximitydriven label transfer. We targeted protein tyrosine kinase 7 (PTK7), a prominent cancer marker, and demonstrated aptamer-mediated biotin transfer to specific lysine residues on the extracellular domain of the protein. This allowed for tracking of PTK7 expression, localization, and cellular internalization. These studies validate the programmability of covalent aptamers and highlight their applicability in a cellular context, including protein and small molecule delivery.

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.

A wide array of optogenetic tools is available that allow for precise spatiotemporal control over many cellular processes. These tools have been especially popular among zebrafish researchers who take advantage of the embryo's transparency. However, photocleavable optogenetic proteins have not been utilized in zebrafish. We demonstrate successful optical control of protein cleavage in embryos using PhoCl, a photocleavable fluorescent protein. This optogenetic tool offers temporal and spatial control over protein cleavage events, which we demonstrate in light-triggered protein translocation and apoptosis.

A wide array of optogenetic tools are available that allow for precise spatiotemporal control over cellular processes. These tools are particularly important to zebrafish researchers who take advantage of the embryo's transparency. However, photocleavable optogenetic proteins have not been utilized in zebrafish. We demonstrate successful optical control of protein cleavage in embryos using PhoCl, a photocleavable fluorescent protein. This optogenetic tool offers temporal and spatial control over protein cleavage events, which we demonstrate in light-triggered protein translocation and light-triggered apoptosis.