Direct Activation of Nucleobases with Small Molecules for the Conditional Control of Antisense Function.

Conditionally controlled antisense oligonucleotides provide precise interrogation of gene function at different developmental stages in animal models. Only one example of small molecule-induced activation of antisense function exist. This has been restricted to cyclic caged morpholinos that, based on sequence, can have significant background activity in the absence of the trigger. Here, we provide a new approach using azido-caged nucleobases that are site-specifically introduced into antisense morpholinos. The caging group design is a simple azidomethylene (Azm) group that, despite its very small size, efficiently blocks Watson-Crick base pairing in a programmable fashion. Furthermore, it undergoes facile decaging via Staudinger reduction when exposed to a small molecule phosphine, generating the native antisense oligonucleotide under conditions compatible with biological environments. We demonstrated small molecule-induced gene knockdown in mammalian cells, zebrafish embryos, and frog embryos. We validated the general applicability of this approach by targeting three different genes.

DNA Logic Gates for Small Molecule Activation Circuits in Cells.

DNA-based devices such as DNA logic gates self-assemble into supramolecular structures, as dictated by the sequences of the constituent oligonucleotides and their predictable Watson-Crick base pairing interactions. The programmable nature of DNA-based devices permits the design and implementation of DNA circuits that interact in a dynamic and sequential manner capable of spatially arranging disparate DNA species. Here, we report the application of an activatable fluorescence reporter based on a proximity-driven inverse electron demand Diels-Alder (IEDDA) reaction and its robust integration with DNA strand displacement circuits. In response to specific DNA input patterns, sequential strand displacement reactions are initiated and culminate in the hybridization of two modified DNA strands carrying probes capable of undergoing an IEDDA reaction between a vinyl-ether-caged fluorophore and its reactive partner tetrazine, leading to the activation of fluorescence. This approach provides a major advantage for DNA computing in mammalian cells since circuit degradation does not induce fluorescence, in contrast to traditional fluorophore-quencher designs. We demonstrate the robustness and sensitivity of the reporter by testing its ability to serve as a readout for DNA logic circuits of varying complexity inside cells.

Quantitative Analysis and Optimization of Site-Specific Protein Bioconjugation in Mammalian Cells.

Despite a range of covalent protein modifications, few techniques exist for quantification of protein bioconjugation in cells. Here, we describe a novel method for quantifying in cellulo protein bioconjugation through covalent bond formation with HaloTag. This approach utilizes unnatural amino acid (UAA) mutagenesis to selectively install a small and bioorthogonally reactive handle onto the surface of a protein. We utilized the fast kinetics and high selectivity of inverse electron-demand Diels-Alder cycloadditions to evaluate reactions of tetrazine phenylalanine (TetF) with strained trans-cyclooctene-chloroalkane (sTCO-CA) and trans-cyclooctene lysine (TCOK) with tetrazine-chloroalkane (Tet-CA). Following bioconjugation, the chloroalkane ligand is exposed for labeling by the HaloTag enzyme, allowing for straightforward quantification of bioconjugation via simple western blot analysis. We demonstrate the versatility of this tool for quickly and accurately determining the bioconjugation efficiency of different UAA/chloroalkane pairs and for different sites on different proteins of interest, including EGFP and the estrogen-related receptor ERRα.

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.

DNA Computing: NOT Logic Gates See the Light.

DNA-based Boolean logic gates (for example, AND, OR, and NOT) can be assembled into complex computational circuits that generate an output signal in response to specific patterns of oligonucleotide inputs. However, the fundamental nature of NOT gates, which convert the absence of an input into an output, makes their implementation within DNA-based circuits difficult. Premature execution of a NOT gate before completion of its upstream computation introduces an irreversible error into the circuit. By utilizing photocaging groups, we developed a novel DNA gate design that prevents gate function until irradiation at a certain time point. Optical activation provides temporal control over circuit performance by preventing premature computation and is orthogonal to all other components of DNA computation devices. Using this approach, we designed NAND and NOR logic gates that respond to synthetic microRNA sequences. We further demonstrate the utility of the NOT gate within multilayer circuits in response to a specific pattern of four microRNAs.

Conditional gene knockdowns in sea urchins using caged morpholinos.

Echinoderms are important experimental models for analyzing embryonic development, but a lack of spatial and temporal control over gene perturbations has hindered developmental studies using these animals. Morpholino antisense oligonucleotides (MOs) have been used successfully by the echinoderm research community for almost two decades, and MOs remain the most widely used tool for acute gene knockdowns in these organisms. Echinoderm embryos develop externally and are optically transparent, making them ideally-suited to many light-based approaches for analyzing and manipulating development. Studies using zebrafish embryos have demonstrated the effectiveness of photoactivatable (caged) MOs for conditional gene knockdowns. Here we show that caged MOs, synthesized using nucleobase-caged monomers, provide light-regulated control over gene expression in sea urchin embryos. Our work provides the first robust approach for conditional gene silencing in this prominent model system.

Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA.

We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5'-protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA-target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off-to-on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.

Development of photolabile protecting groups and their application to the optochemical control of cell signaling.

Many biological processes are naturally regulated with spatiotemporal control. In order to perturb and investigate them, optochemical tools have been developed that convey similar spatiotemporal precision. Pivotal to optochemical probes are photolabile protecting groups, so called caging groups, and recent developments have enabled new applications to cellular processes, including cell signaling. This review focuses on the advances made in the field of caging groups and their application in cell signaling through caged molecules such as neurotransmitters, lipids, secondary messengers, and proteins.