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.

Expanding the Genetic Code of Xenopus laevis Embryos.

The incorporation of unnatural amino acids into proteins through genetic code expansion has been successfully adapted to African claw-toed frog embryos. Six unique unnatural amino acids are incorporated site-specifically into proteins and demonstrate robust and reliable protein expression. Of these amino acids, several are caged analogues that can be used to establish conditional control over enzymatic activity. Using light or small molecule triggers, we exhibit activation and tunability of protein functions in live embryos. This approach was then applied to optical control over the activity of a RASopathy mutant of NRAS, taking advantage of generating explant cultures from Xenopus. Taken together, genetic code expansion is a robust approach in the Xenopus model to incorporate novel chemical functionalities into proteins of interest to study their function and role in a complex biological setting.

Genetically Encoded Aminocoumarin Lysine for Optical Control of Protein-Nucleotide Interactions in Zebrafish Embryos.

The strategic placement of unnatural amino acids into the active site of kinases and phosphatases has allowed for the generation of photocaged signaling proteins that offer spatiotemporal control over activation of these pathways through precise light exposure. However, deploying this technology to study cell signaling in the context of embryo development has been limited. The promise of optical control is especially useful in the early stages of an embryo where development is driven by tightly orchestrated signaling events. Here, we demonstrate light-induced activation of Protein Kinase A and a RASopathy mutant of NRAS in the zebrafish embryo using a new light-activated amino acid. We applied this approach to gain insight into the roles of these proteins in gastrulation and heart development and forge a path for further investigation of RASopathy mutant proteins in animals.

Small-Molecule Phosphine Activation of Protein Function in Zebrafish Embryos with an Expanded Genetic Code.

Conditional control of protein function in a living model organism is an important tool for studying the effects of that protein during development and disease. In this chapter, we walk through the steps to generate a small-molecule-activatable enzyme in zebrafish embryos through the incorporation of a noncanonical amino acid into the protein active site. This method can be applied to many enzyme classes, which we highlight with temporal control of a luciferase and a protease. We demonstrate that strategic placement of the noncanonical amino acid completely blocks enzyme activity, which is then promptly restored after addition of the nontoxic small molecule inducer to the embryo water.

Engineering Small Molecule Switches of Protein Function in Zebrafish Embryos.

Precise temporally regulated protein function directs the highly complex processes that make up embryo development. The zebrafish embryo is an excellent model organism to study development, and conditional control over enzymatic activity is desirable to target chemical intervention to specific developmental events and to investigate biological mechanisms. Surprisingly few, generally applicable small molecule switches of protein function exist in zebrafish. Genetic code expansion allows for site-specific incorporation of unnatural amino acids into proteins that contain caging groups that are removed through addition of small molecule triggers such as phosphines or tetrazines. This broadly applicable control of protein function was applied to activate several enzymes, including a GTPase and a protease, with temporal precision in zebrafish embryos. Simple addition of the small molecule to the media produces robust and tunable protein activation, which was used to gain insight into the development of a congenital heart defect from a RASopathy mutant of NRAS and to control DNA and protein cleavage events catalyzed by a viral recombinase and a viral protease, respectively.

Chemically Acylated tRNAs are Functional in Zebrafish Embryos.

Genetic code expansion has pushed protein chemistry past the canonical 22 amino acids. The key enzymes that make this possible are engineered aminoacyl tRNA synthetases. However, as the number of genetically encoded amino acids has increased over the years, obvious limits in the type and size of novel side chains that can be accommodated by the synthetase enzyme become apparent. Here, we show that chemically acylating tRNAs allow for robust, site-specific incorporation of unnatural amino acids into proteins in zebrafish embryos, an important model organism for human health and development. We apply this approach to incorporate a unique photocaged histidine analogue for which synthetase engineering efforts have failed. Additionally, we demonstrate optical control over different enzymes in live embryos by installing photocaged histidine into their active sites.

Optogenetic Protein Cleavage in Zebrafish Embryos.

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.

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.

Regulating CRISPR/Cas9 Function through Conditional Guide RNA Control.

Conditional control of CRISPR/Cas9 has been developed by using a variety of different approaches, many focusing on manipulation of the Cas9 protein itself. However, more recent strategies for governing CRISPR/Cas9 function are based on guide RNA (gRNA) modifications. They include control of gRNAs by light, small molecules, proteins, and oligonucleotides. These designs have unique advantages compared to other approaches and have allowed precise regulation of gene editing and transcription. Here, we discuss strategies for conditional control of gRNA function and compare effectiveness of these methods.

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.

Light-activation of Cre recombinase in zebrafish embryos through genetic code expansion.

Cre recombinase-mediated DNA recombination is an established method for conditional control of gene expression in animal models. Regulation of its activity has been accomplished to impart spatial and/or temporal control over recombination of the target gene. In this chapter, optical control of Cre recombinase in developing zebrafish embryos through genetic code expansion is discussed. This method takes advantage of an evolved aminoacyl tRNA synthetase and tRNA pair that can incorporate an unnatural amino acid (UAA) into proteins in response to an amber stop codon (TAG). Genetic code expansion is used to replace a lysine residue critical to Cre recombinase function with a photocaged analogue of lysine, successfully blocking DNA recombination until irradiation with 405nm light. Use of optically controlled Cre recombinase for cell-lineage tracing experiments in zebrafish embryos is highlighted, demonstrating the ability to target small populations of cells at different developmental time points for recombination. Optically controlled Cre recombinase showed no background activity and precise activation upon irradiation, making it a useful new tool for studying development and disease in the zebrafish embryo.

Genetic Code Expansion in Animals.

Expanding the genetic code to enable the incorporation of unnatural amino acids into proteins in biological systems provides a powerful tool for studying protein structure and function. While this technology has been mostly developed and applied in bacterial and mammalian cells, it recently expanded into animals, including worms, fruit flies, zebrafish, and mice. In this review, we highlight recent advances toward the methodology development of genetic code expansion in animal model organisms. We further illustrate the applications, including proteomic labeling in fruit flies and mice and optical control of protein function in mice and zebrafish. We summarize the challenges of unnatural amino acid mutagenesis in animals and the promising directions toward broad application of this emerging technology.

Cell-Lineage Tracing in Zebrafish Embryos with an Expanded Genetic Code.

Cell-lineage tracing is used to study embryo development and stem-cell differentiation as well as to document tumor cell heterogeneity. Cre recombinase-mediated cell labeling is the preferred approach; however, its utility is restricted by when and where DNA recombination takes place. We generated a photoactivatable Cre recombinase by replacing a critical residue in its active site with a photocaged lysine derivative through genetic code expansion in zebrafish embryos. This allows high spatiotemporal control of DNA recombination by using 405 nm irradiation. Importantly, no background activity is seen before irradiation, and, after light-triggered removal of the caging group, Cre recombinase activity is restored. We demonstrate the utility of this tool as a cell-lineage tracer through its activation in different regions and at different time points in the early embryo. Direct control of Cre recombinase by light will allow more precise DNA recombination, thereby enabling more nuanced studies of metazoan development and disease.

Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid.

Bioorthogonal reactions for labeling biomolecules in live cells have been limited by slow reaction rates or low component selectivity and stability. Ideal bioorthogonal reactions with high reaction rates, high selectivity, and high stability would allow for stoichiometric labeling of biomolecules in minutes and eliminate the need to wash out excess labeling reagent. Currently, no general method exists for controlled stoichiometric or substoichiometric labeling of proteins in live cells. To overcome this limitation, we developed a significantly improved tetrazine-containing amino acid (Tet-v2.0) and genetically encoded Tet-v2.0 with an evolved aminoacyl-tRNA synthetase/tRNA(CUA) pair. We demonstrated in cellulo that protein containing Tet-v2.0 reacts selectively with cyclopropane-fused trans-cyclooctene (sTCO) with a bimolecular rate constant of 72,500 ± 1660 M(-1) s(-1) without reacting with other cellular components. This bioorthogonal ligation of Tet-v2.0-protein reacts in cellulo with substoichiometric amounts of sTCO-label fast enough to remove the labeling reagent from media in minutes, thereby eliminating the need to wash out label. This ideal bioorthogonal reaction will enable the monitoring of a larger window of cellular processes in real time.

Liquid chromatography/tandem mass spectrometry sensitivity enhancement via online sample dilution and trapping: applications in microdosing and dried blood spot (DBS) bioanalysis.

A simple online sample dilution, enrichment, and cleanup technique was developed for sensitive microdosing and dried blood spot (DBS) liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalysis. Samples are diluted online with water and enriched in a trap column which is subsequently switched inline with the analytical column. Excellent lansoprazole (in acetonitrile) peak shape is maintained even with an 80-microL injection. In comparison, similar chromatographic peaks were observed only when a small volume of the same solution, i.e., 1 microL, was injected on a regular high-performance liquid chromatography (HPLC) system, where an injection of 5 microL resulted in severe peak fronting. A substantial enhancement in sensitivity is realized in the trapping mode using large injection volumes. The trap column is washed at the beginning and at the end of each injection with aqueous and organic solvent respectively to remove matrix components. This ultimately leads to reduction of matrix effects and mass spectrometer noise, thus facilitating the utilization of protein precipitation as the sample preparation for plasma samples. A lower limit of quantitation (LLOQ) of 0.5 pg/mL was demonstrated for lansoprazole in human plasma with a signal-to-noise (S/N) ratio of 13 using a 100 microL injection. Excellent intra-day precision and accuracy were established for lansoprazole in human plasma with good linearity (R(2) > 0.999) from 0.5 to 500 pg/mL. This level of LLOQ makes LC/MS/MS a practical alternative for microdosing bioanalysis, where the dose is typically 100 times lower than the therapeutic dose. The same technique was applied to quantitate lansoprazole in human whole blood employing DBS technology. With a single 3-mm punch, i.e. approximately 2 microL of whole blood or approximately 1 microL plasma, a LLOQ of 0.1 ng/mL showed sufficient S/N ratio (40) for lansoprazole when 75 microL of extract was injected. In all, the online sample dilution, cleanup, and enrichment technique demonstrated the practical utility of LC/MS/MS in microdosing and DBS bioanalysis.