Antisense Oligonucleotide Activation via Enzymatic Antibiotic Resistance Mechanism.

The structure and mechanism of the bacterial enzyme ß-lactamase have been well-studied due to its clinical role in antibiotic resistance. ß-Lactamase is known to hydrolyze the ß-lactam ring of the cephalosporin scaffold, allowing a spontaneous self-immolation to occur. Previously, cephalosporin-based sensors have been developed to evaluate ß-lactamase expression in both mammalian cells and zebrafish embryos. Here, we present a circular caged morpholino oligonucleotide (cMO) activated by ß-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 ß-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 ß-lactamase to the current suite of enzymatic triggers presents unique opportunities for robust, orthogonal control over endogenous gene expression in a spatially resolved manner.

Translational control of gene function through optically regulated nucleic acids.

Translation of mRNA into protein is one of the most fundamental processes within biological systems. Gene expression is tightly regulated both in space and time, often involving complex signaling or gene regulatory networks, as most prominently observed in embryo development. Thus, studies of gene function require tools with a matching level of external control. Light is an excellent conditional trigger as it is minimally invasive, can be easily tuned in wavelength and amplitude, and can be applied with excellent spatial and temporal resolution. To this end, modification of established oligonucleotide-based technologies with optical control elements, in the form of photocaging groups and photoswitches, has rendered these tools capable of navigating the dynamic regulatory pathways of mRNA translation in cellular and in vivo models. In this review, we discuss the different optochemical approaches used to generate photoresponsive nucleic acids that activate and deactivate gene expression and function at the translational level.

Blue Light Activated Rapamycin for Optical Control of Protein Dimerization in Cells and Zebrafish Embryos.

Rapamycin-induced dimerization of FKBP and FRB is the most commonly utilized chemically induced protein dimerization system. It has been extensively used to conditionally control protein localization, split-enzyme activity, and protein-protein interactions in general by simply fusing FKBP and FRB to proteins of interest. We have developed a new aminonitrobiphenylethyl caging group and applied it to the generation of a caged rapamycin analog that can be photoactivated using blue light. Importantly, the caged rapamycin analog shows minimal background activity with regard to protein dimerization and can be directly interfaced with a wide range of established (and often commercially available) FKBP/FRB systems. We have successfully demonstrated its applicability to the optical control of enzymatic function, protein stability, and protein subcellular localization. Further, we also showcased its applicability toward optical regulation of cell signaling, specifically mTOR signaling, in cells and aquatic embryos.