ABSTRACT
Site-specific incorporation of unnatural amino acids into proteins provides a powerful tool to study protein function. Here we report genetic code expansion in zebrafish embryos and its application to the optogenetic control of cell signaling. We genetically encoded four unnatural amino acids with a diverse set of functional groups, which included a photocaged lysine that was applied to the light-activation of luciferase and kinase activity. This approach enables versatile manipulation of protein function in live zebrafish embryos, a transparent and commonly used model organism to study embryonic development.
Subject(s)
Amino Acids/genetics , Light , Luciferases/genetics , Phosphotransferases/genetics , Signal Transduction , Amino Acids/metabolism , Animals , Genetic Code , Luciferases/metabolism , Phosphotransferases/metabolism , Zebrafish/embryology , Zebrafish/metabolismABSTRACT
The CRISPR/Cas9 system has emerged as an important tool in biomedical research for a wide range of applications, with significant potential for genome engineering and gene therapy. In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid. Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function.
Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genetic Engineering , RNA Editing/genetics , HEK293 Cells , Humans , Lysine/chemistry , Lysine/genetics , Models, Molecular , Molecular StructureABSTRACT
The spliceosome machinery is composed of several proteins and multiple small RNA molecules that are involved in gene regulation through the removal of introns from pre-mRNAs in order to assemble exon-based mRNA containing protein-coding sequences. Splice-switching oligonucleotides (SSOs) are genetic control elements that can be used to specifically control the expression of genes through correction of aberrant splicing pathways. A current limitation with SSO methodologies is the inability to achieve conditional control of their function paired with high spatial and temporal resolution. We addressed this limitation through site-specific installation of light-removable nucleobase-caging groups as well as photocleavable backbone linkers into synthetic SSOs. This enables optochemical OFF â ON and ON â OFF switching of their activity and thus precise control of alternative splicing. The use of light as a regulatory element allows for tight spatial and temporal control of splice switching in mammalian cells and animals.
Subject(s)
Alternative Splicing/radiation effects , Light , Oligonucleotides/genetics , Animals , HeLa Cells , Humans , Oligonucleotides/chemistry , ZebrafishABSTRACT
The hybridization chain reaction (HCR) and fuel-catalyst cycles have been applied to address the problem of signal amplification in DNA-based computation circuits. While they function efficiently, these signal amplifiers cannot be switched ON or OFF quickly and noninvasively. To overcome these limitations, a light-activated initiator strand for the HCR, which enabled fast optical OFF â ON switching, was developed. Similarly, when a light-activated version of the catalyst strand or the inhibitor strand of a fuel-catalyst cycle was applied, the cycle could be optically switched from OFF â ON or ON â OFF, respectively. To move the capabilities of these devices beyond solution-based operations, the components were embedded in agarose gels. Irradiation with customizable light patterns and at different time points demonstrated both spatial and temporal control. The addition of a translator gate enabled a spatially activated signal to travel along a predefined path, akin to a chemical wire. Overall, the addition of small light-cleavable photocaging groups to DNA signal amplification circuits enabled conditional control as well as fast photocontrol of signal amplification.
Subject(s)
Computers, Molecular , DNA/chemistry , Catalysis , Nucleic Acid Amplification Techniques , Nucleic Acid Hybridization , Photochemistry/methodsABSTRACT
In cell and molecular biology, double-stranded circular DNA constructs, known as plasmids, are extensively used to express a gene of interest. These gene expression systems rely on a specific promoter region to drive the transcription of genes either constitutively (i.e., in a continually "ON" state) or conditionally (i.e., in response to a specific transcription initiator). However, controlling plasmid-based expression with high spatial and temporal resolution in cellular environments and in multicellular organisms remains challenging. To overcome this limitation, we have site-specifically installed nucleobase-caging groups within a plasmid promoter region to enable optochemical control of transcription and, thus, gene expression, via photolysis of the caging groups. Through the light-responsive modification of plasmid-based gene expression systems, we have demonstrated optochemical activation of an exogenous fluorescent reporter gene in both tissue culture and a live animal model, as well as light-induced overexpression of an endogenous signaling protein.
Subject(s)
DNA, Circular/genetics , Plasmids/genetics , Promoter Regions, Genetic , Transcriptional Activation , Animals , Base Sequence , DNA, Circular/chemistry , Green Fluorescent Proteins/genetics , HEK293 Cells , Humans , Molecular Sequence Data , Photolysis , Plasmids/chemistry , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Proteins , Ultraviolet Rays , ZebrafishABSTRACT
The migration of cortical projection neurons is a multistep process characterized by dynamic cell shape remodeling. The molecular basis of these changes remains elusive, and the present work describes how microRNAs (miRNAs) control neuronal polarization during radial migration. We show that miR-22 and miR-124 are expressed in the cortical wall where they target components of the CoREST/REST transcriptional repressor complex, thereby regulating doublecortin transcription in migrating neurons. This molecular pathway underlies radial migration by promoting dynamic multipolar-bipolar cell conversion at early phases of migration, and later stabilization of cell polarity to support locomotion on radial glia fibers. Thus, our work emphasizes key roles of some miRNAs that control radial migration during cerebral corticogenesis.
Subject(s)
Cell Movement/physiology , Cerebral Cortex/cytology , MicroRNAs/metabolism , Nerve Tissue Proteins/metabolism , Neurons/cytology , Repressor Proteins/metabolism , Animals , Cell Polarity/physiology , Cells, Cultured , Co-Repressor Proteins , Mice , Mice, Transgenic , Neurons/metabolismABSTRACT
Photocaging provides a method to spatially and temporally control biological function and gene expression with high resolution. Proteins can be photochemically controlled through the site-specific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochemical activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference.
Subject(s)
Activating Transcription Factors/genetics , DNA-Directed RNA Polymerases/genetics , Gene Expression Regulation, Enzymologic/genetics , Gene Silencing , Light , Viral Proteins/genetics , Activating Transcription Factors/metabolism , DNA-Directed RNA Polymerases/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Models, Molecular , Molecular Structure , Photochemical Processes , Protein Engineering , Viral Proteins/metabolismABSTRACT
DNA computation can utilize logic gates as modules to create molecular computers with biological inputs. Modular circuits that recognize nucleic acid inputs through strand hybridization activate computation cascades to produce controlled outputs. This allows for the construction of synthetic circuits that can be interfaced with cellular environments. We have engineered oligonucleotide AND gates to respond to specific microRNA (miRNA) inputs in live mammalian cells. Both single and dual-sensing miRNA-based computation devices were synthesized for the cell-specific identification of endogenous miR-21 and miR-122. A logic gate response was observed with miRNA expression regulators, exhibiting molecular recognition of miRNA profile changes. Nucleic acid logic gates that are functional in a cellular environment and recognize endogenous inputs significantly expand the potential of DNA computation to monitor, image, and respond to cell-specific markers.
Subject(s)
Computers, Molecular , DNA/chemistry , MicroRNAs/chemistry , DNA/metabolism , HeLa Cells , Humans , MicroRNAs/metabolism , Photochemical ProcessesABSTRACT
MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional gene regulators and have been shown to regulate many biological processes including embryonal development, cell differentiation, apoptosis, and proliferation. Variations in the expression of certain miRNAs have been linked to a wide range of human diseases - especially cancer - and the diversity of miRNA targets suggests that they are involved in various cellular networks. Several tools have been developed to control the function of individual miRNAs and have been applied to study their biogenesis, biological role, and therapeutic potential; however, common methods lack a precise level of control that allows for the study of miRNA function with high spatial and temporal resolution. Light-activated miRNA antagomirs for mature miR-122 and miR-21 were developed through the site-specific installation of caging groups on the bases of selected nucleotides. Installation of caged nucleotides led to complete inhibition of the antagomir-miRNA hybridization and thus inactivation of antagomir function. The miRNA-inhibitory activity of the caged antagomirs was fully restored upon decaging through a brief UV irradiation. The synthesized antagomirs were applied to the photochemical regulation of miRNA function in mammalian cells. Moreover, spatial control over antagomir activity was obtained in mammalian cells through localized UV exposure. The presented approach enables the precise regulation of miRNA function and miRNA networks with unprecedented spatial and temporal resolution using UV irradiation and can be extended to any miRNA of interest.
Subject(s)
Light , MicroRNAs/antagonists & inhibitors , MicroRNAs/radiation effects , Oligonucleotides/pharmacology , Oligonucleotides/radiation effects , Antagomirs , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Differentiation/drug effects , Cell Differentiation/radiation effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Humans , MicroRNAs/metabolism , Oligonucleotides/chemistry , Photochemistry , Ultraviolet RaysABSTRACT
Triplex-forming oligonucleotides (TFOs) are efficient tools to regulate gene expression through the inhibition of transcription. Here, nucleobase-caging technology was applied to the temporal regulation of transcription through light-activated TFOs. Through site-specific incorporation of caged thymidine nucleotides, the TFO:DNA triplex formation is blocked, rendering the TFO inactive. However, after a brief UV irradiation, the caging groups are removed, activating the TFO and leading to the inhibition of transcription. Furthermore, the synthesis and site-specific incorporation of caged deoxycytidine nucleotides within TFO inhibitor sequences was developed, allowing for the light-deactivation of TFO function and thus photochemical activation of gene expression. After UV-induced removal of the caging groups, the TFO forms a DNA dumbbell structure, rendering it inactive, releasing it from the DNA, and activating transcription. These are the first examples of light-regulated TFOs and their application in the photochemical activation and deactivation of gene expression. In addition, hairpin loop structures were found to significantly increase the efficacy of phosphodiester DNA-based TFOs in tissue culture.
Subject(s)
DNA/metabolism , Nucleic Acid Conformation , Oligonucleotides/metabolism , Photic Stimulation , Transcription, Genetic/physiology , DNA/chemistry , HEK293 Cells , Humans , Oligonucleotides/chemistry , Photic Stimulation/methodsABSTRACT
DNA computation is an emerging field that enables the assembly of complex circuits based on defined DNA logic gates. DNA-based logic gates have previously been operated through purely chemical means, controlling logic operations through DNA strands or other biomolecules. Although gates can operate through this manner, it limits temporal and spatial control of DNA-based logic operations. A photochemically controlled AND gate was developed through the incorporation of caged thymidine nucleotides into a DNA-based logic gate. By using light as the logic inputs, both spatial control and temporal control were achieved. In addition, design rules for light-regulated DNA logic gates were derived. A step-response, which can be found in a controller, was demonstrated. Photochemical inputs close the gap between DNA computation and silicon-based electrical circuitry, since light waves can be directly converted into electrical output signals and vice versa. This connection is important for the further development of an interface between DNA logic gates and electronic devices, enabling the connection of biological systems with electrical circuits.