The human Shu complex promotes RAD51 activity by modulating RPA dynamics on ssDNA.

Templated DNA repair that occurs during homologous recombination and replication stress relies on RAD51. RAD51 activity is positively regulated by BRCA2 and the RAD51 paralogs. The Shu complex is a RAD51 paralog-containing complex consisting of SWSAP1 and SWS1. We demonstrate that SWSAP1-SWS1 binds RAD51, maintains RAD51 filament stability, and enables strand exchange. Using single molecule confocal fluorescence microscopy combined with optical tweezers, we show that SWSAP1-SWS1 decorates RAD51 filaments proficient for homologous recombination. We also find SWSAP1-SWS1 enhances RPA diffusion on ssDNA. Importantly, we show human sgSWSAP1 and sgSWS1 knockout cells are sensitive to pharmacological inhibition of PARP and APE1. Lastly, we identify cancer variants in SWSAP1 that alter SWS1 complex formation. Together, we show that SWSAP1-SWS1 stimulates RAD51-dependent high-fidelity repair and may be an important new cancer therapeutic target.

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.

Homologous recombination-deficient mutation cluster in tumor suppressor RAD51C identified by comprehensive analysis of cancer variants.

Mutations in homologous recombination (HR) genes, including BRCA1, BRCA2, and the RAD51 paralog RAD51C, predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes.

Optical Control of MicroRNA Function in Zebrafish Embryos.

MicroRNAs play crucial and dynamic roles in vertebrate development and diseases. Some, like miR-430, are highly expressed during early embryo development and regulate hundreds of transcripts, which can make it difficult to study their role in the timing and location of specific developmental processes using conventional morpholino oligonucleotide (MO) knockdown or genetic deletion approaches. We demonstrate that light-activated circular morpholino oligonucleotides (cMOs) can be applied to the conditional control of microRNA function. We targeted miR-430 in zebrafish embryos to study its role in the development of the embryo body and the heart. Using 405 nm irradiation, precise spatial and temporal control over miR-430 function was demonstrated, offering insight into the cell populations and developmental timepoints involved in each process.

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.

Small Molecule Control of Morpholino Antisense Oligonucleotide Function through Staudinger Reduction.

Conditionally activated, caged morpholino antisense agents (cMOs) are tools that enable the temporal and spatial investigation of gene expression, regulation, and function during embryonic development. Cyclic MOs are conformationally gated oligonucleotide analogs that do not block gene expression until they are linearized through the application of an external trigger, such as light or enzyme activity. Here, we describe the first examples of small molecule-responsive cMOs, which undergo rapid and efficient decaging via a Staudinger reduction. This is enabled by a highly flexible linker design that offers opportunities for the installation of chemically activated, self-immolative motifs. We synthesized cyclic cMOs against two distinct, developmentally relevant genes and demonstrated phosphine-triggered knockdown of gene expression in zebrafish embryos. This represents the first report of a small molecule-triggered antisense agent for gene knockdown, adding another bioorthogonal entry to the growing arsenal of gene knockdown tools.

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.

Small-molecule control of neurotransmitter sulfonation.

Controlling unmodified serotonin levels in brain synapses is a primary objective when treating major depressive disorder-a disease that afflicts ∼20% of the world's population. Roughly 60% of patients respond poorly to first-line treatments and thus new therapeutic strategies are sought. To this end, we have constructed isoform-specific inhibitors of the human cytosolic sulfotransferase 1A3 (SULT1A3)-the isoform responsible for sulfonating ∼80% of the serotonin in the extracellular brain fluid. The inhibitor design includes a core ring structure, which anchors the inhibitor into a SULT1A3-specific binding pocket located outside the active site, and a side chain crafted to act as a latch to inhibit turnover by fastening down the SULT1A3 active-site cap. The inhibitors are allosteric, they bind with nanomolar affinity and are highly specific for the 1A3 isoform. The cap-stabilizing effects of the latch can be accurately calculated and are predicted to extend throughout the cap and into the surrounding protein. A free-energy correlation demonstrates that the percent inhibition at saturating inhibitor varies linearly with cap stabilization - the correlation is linear because the rate-limiting step of the catalytic cycle, nucleotide release, scales linearly with the fraction of enzyme in the cap-open form. Inhibitor efficacy in cultured cells was studied using a human mammary epithelial cell line that expresses SULT1A3 at levels comparable with those found in neurons. The inhibitors perform similarly in ex vivo and in vitro studies; consequently, SULT1A3 turnover can now be potently suppressed in an isoform-specific manner in human cells.

Optical Control of Small Molecule-Induced Protein Degradation.

As an emerging approach to protein perturbation, small molecule-induced protein degradation has gained significant attention as both a chemical tool and a potential therapeutic. To enable discrete control over its function, we have developed a broadly applicable approach for the optical activation of small molecule-induced protein degradation. By installing two different photolabile protecting groups, so-called caging groups, onto two different ligands recruiting Von Hippel-Lindau (VHL) and cereblon (CRBN) E3 ubiquitin ligases, our strategy enables light-triggered protein degradation for any small molecule warhead.

Allosteres to regulate neurotransmitter sulfonation.

Catecholamine neurotransmitter levels in the synapses of the brain shape human disposition-cognitive flexibility, aggression, depression, and reward seeking-and manipulating these levels is a major objective of the pharmaceutical industry. Certain neurotransmitters are extensively sulfonated and inactivated by human sulfotransferase 1A3 (SULT1A3). To our knowledge, sulfonation as a therapeutic means of regulating transmitter activity has not been explored. Here, we describe the discovery of a SULT1A3 allosteric site that can be used to inhibit the enzyme. The structure of the new site is determined using spin-label-triangulation NMR. The site forms a cleft at the edge of a conserved ∼30-residue active-site cap that must open and close during the catalytic cycle. Allosteres anchor into the site via π-stacking interactions with two residues that sandwich the planar core of the allostere and inhibit the enzyme through cap-stabilizing interactions with substituents attached to the core. Changes in cap free energy were calculated ab initio as a function of core substituents and used to design and synthesize a series of inhibitors intended to progressively stabilize the cap and slow turnover. The inhibitors bound tightly (34 nm to 7.4 µm) and exhibited progressive inhibition. The cap-stabilizing effects of the inhibitors were experimentally determined and agreed remarkably well with the theoretical predictions. These studies establish a reliable heuristic for the design of SULT1A3 allosteric inhibitors and demonstrate that the free-energy changes of a small, dynamic loop that is critical for SULT substrate selection and turnover can be calculated accurately.