Oligonucleotides are effective tools for the regulation of gene expression in cell culture and model organisms, most importantly through antisense mechanisms. Due to the inherent instability of DNA antisense agents, various modifications have been introduced to increase the efficacy of oligonucleotides, including phosphorothioate DNA, locked nucleic acids, peptide nucleic acids, and others. Here, we present antisense agent stabilization through conjugation of a polyethylene glycol (PEG) group to a DNA oligonucleotide. By employing a photocleavable linker between the PEG group and the antisense agent we were able to achieve light-induced deactivation of antisense activity. The bioconjugated PEG group provides stability to the DNA antisense agent without affecting its native function of silencing gene expression via RNase H-catalyzed messenger RNA degradation. Once irradiated with UV light of 365 nm, the PEG group is cleaved from the antisense agent leaving the DNA unprotected and open for degradation by endogenous nucleases, thereby restoring gene expression. By using a photocleavable PEG group (PhotoPEG), antisense activity can be regulated with high spatial and temporal resolution, paving the way for precise regulation of gene expression in biological systems.

DNA decoys have been developed for the inhibition of the transcriptional regulation of gene expression. However, the present methodology lacks the spatial and temporal control of gene expression that is commonly found in nature. Here, we report the application of photo-removable protecting groups on nucleobases of NF-κB DNA decoys to regulate NF-κB driven transcription of secreted alkaline phosphatase using light as an external control element. The NF-κB family of proteins is comprised of important eukaryotic transcription factors that regulate a wide range of cellular processes and are involved in immune response, development, cellular growth, and cell death. Several diseases, including cancer, arthritis, chronic inflammation, asthma, neurodegenerative diseases, and heart disease have been linked to constitutively active NF-κB. Through the direct incorporation of caging groups into an NF-κB decoy we were able to disrupt DNA:DNA hybridization and inhibit the binding of the transcription factor to the DNA decoy until UV irradiation removes the caging groups and restores the activity of the oligonucleotide. Excellent light-switching behavior of transcriptional regulation was observed. This is the first example of a caged DNA decoy for the photochemical regulation of gene expression in mammalian cells and represents an important addition to the toolbox of light-controlled gene regulatory agents.

We developed a new system for light-induced protein dimerization in living cells using a novel photocaged analog of rapamycin (pRap) together with an engineered rapamycin binding domain (iFKBP). Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo.

Summary

Disruptions of anatomical left-right asymmetry result in life-threatening heterotaxic birth defects in vital organs. We performed a small molecule screen for left-right asymmetry phenotypes in Xenopus embryos and discovered a novel pyridine analog, heterotaxin, which disrupts both cardiovascular and digestive organ laterality and inhibits TGF-β-dependent left-right asymmetric gene expression. Heterotaxin analogs also perturb vascular development, melanogenesis, cell migration and adhesion, and indirectly inhibit the phosphorylation of an intracellular mediator of TGF-β signaling. This combined phenotypic profile identifies these compounds as a novel class of TGF-β signaling inhibitors. Notably, heterotaxin analogs also possess highly desirable anti-tumor properties, inhibiting epithelial-mesenchymal transition, angiogenesis and tumor cell proliferation in mammalian systems. Our results suggest that assessing multiple organ, tissue, cellular and molecular parameters in a whole organism context is a valuable strategy for identifying the mechanism of action of novel compounds.

Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue specific gene disruptions in embryos. In addition, current indirect methods for spatiotemporal regulation of morpholino activity in vivo may have off-target effects. Here we report a novel and efficient synthetic route for directly incorporating photocaged monomeric building blocks into morpholino oligomers, and demonstrate the utility of these caged morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino targeting enhanced green fluorescent protein (EGFP) disrupts EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that directly photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatio-temporal control of gene expression in multiple contexts.

Biological processes are regulated with a high level of spatial and temporal resolution. In order to understand and manipulate these processes, scientists need to be able to regulate them with Nature’s level of precision. In this context, light is a unique regulatory element because it can be precisely controlled in location, timing and amplitude. Moreover, most biological laboratories have a wide range of light sources as standard equipment. This review article summarizes the most recent advances in light-mediated regulation of protein function and the application in a cellular context. Specifically, the photocaging of small molecule modulators of protein function and of select amino acid residues in proteins will be discussed. In addition, examples of the photochemical control of protein function through the application of natural light-receptors are presented.

The photochemical regulation of biological systems represents a very precise means of achieving high-resolution control over gene expression in both a spatial and a temporal fashion. DNAzymes are enzymatically active deoxyoligonucleotides that enable the site-specific cleavage of RNA, and have been used in a variety of in vitro applications. We have previously reported the photochemical activation of DNAzymes and antisense agents through the preparation of a caged DNA phosphoramidite and its site-specific incorporation into oligonucleotides. The presence of the caging group disrupts either DNA:RNA hybridization or catalytic activity, until removed via a brief irradiation with UV light. Here, we are expanding this concept by investigating the photochemical deactivation of DNAzymes and antisense agents. Moreover, we report the application of light-activated and light-deactivated antisense agents to the regulation of gene function in mammalian cells. This represents the first example of gene silencing antisense agents that can be turned on and turned off in mammalian tissue culture.

A new and efficient route to the recently reported 3-nitro-2-ethyldibenzofuran caging group was developed. Furthermore, its installation on a thymidine phosphoramidite is described. This caging group is efficiently removed through light-irradiation at 365 nm.

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway.

SUMMARY OF RECENT ADVANCES

Recently, several advances have been made in the activation and deactivation of gene expression using light. These developments are based on the application of small molecule inducers of gene expression, antisense- or RNA interference-mediated gene silencing, and the photochemical control of proteins regulating gene function. The majority of the examples employ a classical “caging technology”, through the chemical installation of a light-removable protecting group on the biological molecule (small molecule, oligonucleotide, or protein) of interest and rendering it inactive. UV light irradiation then removes the caging group and activates the molecule, enabling control over gene activity with high spatial and temporal resolution.

Recently, the RNA interference (RNAi) pathway has become the target of small molecule inhibitors and activators. RNAi has been well established as a research tool in the sequence-specific silencing of genes in eukaryotic cells and organisms by using exogenous, small, double-stranded RNA molecules of approximately 20 nucleotides. Moreover, a recently discovered post-transcriptional gene regulatory mechanism employs microRNAs (miRNAs), a class of endogenously expressed small RNA molecules, which are processed via the RNAi pathway. The chemical modulation of RNAi has important therapeutic relevance, because a wide range of miRNAs has been linked to a variety of human diseases, especially cancer. Thus, the activation of tumor-suppressive miRNAs and the inhibition of oncogenic miRNAs by small molecules have the potential to provide a fundamentally new approach for the development of cancer therapeutics.

The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids.

Human mitochondrial methionine transfer RNA (hmtRNAMetCAU) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position-34 (f5C34). The role of this modification in (hmtRNAMetCAU) for the decoding of AUA, as well as AUG, in both the peptidyl- and aminoacyl-sites of the ribosome in either chain initiation or chain elongation is still unknown. We report the first synthesis and analyses of the tRNA's anticodon stem and loop domain containing the 5-formylcytidine modification. The modification contributes to the tRNA's anticodon domain structure, thermodynamic properties and its ability to bind codons AUA and AUG in translational initiation and elongation.

A new photocaged nucleoside was synthesized and incorporated into DNA using standard synthesis conditions. This approach enabled the disruption of specific H-bonds and allowed for the analysis of their contribution to the activity of a DNAzyme. Brief irradiation with non-photodamaging UV light led to rapid decaging and almost quantitative restoration of DNAzyme activity. The developed strategy has the potential to find widespread application in the light-induced regulation of oligonucleotide function.

We report the discovery of a simple system through which variant pyrrolysyl-tRNA synthetase/tRNACUAPyl pairs created in Escherichia coli can be used to expand the genetic code of Saccharomyces cerevisiae. In the process we have solved the key challenges of producing a functional tRNACUAPyl in yeast and discovered a pyrrolysyl-tRNA synthetase/tRNACUAPyl pair that is orthogonal in yeast. Using our approach we have incorporated an alkyne-containing amino acid for click chemistry, an important post-translationally modified amino acid and one of its analogs, a photocaged amino acid and a photo-cross-linking amino acid into proteins in yeast. Extensions of our approach will allow the growing list of useful amino acids that have been incorporated in E. coli with variant pyrrolysyl-tRNA synthetase/tRNACUAPyl pairs to be site-specifically incorporated into proteins in yeast.