An optimized chemical-genetic method for cell-specific metabolic labeling of RNA.

Tissues and organs are composed of diverse cell types, which poses a major challenge for cell-type-specific profiling of gene expression. Current metabolic labeling methods rely on exogenous pyrimidine analogs that are only incorporated into RNA in cells expressing an exogenous enzyme. This approach assumes that off-target cells cannot incorporate these analogs.

Expanding the Scope of RNA Metabolic Labeling with Vinyl Nucleosides and Inverse Electron-Demand Diels-Alder Chemistry.

Optimized and stringent chemical methods to profile nascent RNA expression are still in demand. Herein, we expand the toolkit for metabolic labeling of RNA through application of inverse electron demand Diels-Alder (IEDDA) chemistry. Structural examination of metabolic enzymes guided the design and synthesis of vinyl-modified nucleosides, which we systematically tested for their ability to be installed through cellular machinery.

The DNA modification N6-methyl-2'-deoxyadenosine (m6dA) drives activity-induced gene expression and is required for fear extinction.

DNA modification is known to regulate experience-dependent gene expression. However, beyond cytosine methylation and its oxidated derivatives, very little is known about the functional importance of chemical modifications on other nucleobases in the brain. Here we report that in adult mice trained in fear extinction, the DNA modification N6-methyl-2'-deoxyadenosine (m6dA) accumulates along promoters and coding sequences in activated prefrontal cortical neurons.

Bioorthogonal Metabolic Labeling of Nascent RNA in Neurons Improves the Sensitivity of Transcriptome-Wide Profiling.

Transcriptome-wide expression profiling of neurons has provided important insights into the underlying molecular mechanisms and gene expression patterns that transpire during learning and memory formation. However, there is a paucity of tools for profiling stimulus-induced RNA within specific neuronal cell populations. A bioorthogonal method to chemically label nascent (i.

Peptidic inhibitors of insulin-degrading enzyme with potential for dermatological applications discovered via phage display.

Insulin-degrading enzyme (IDE) is an atypical zinc-metalloendopeptidase that hydrolyzes insulin and other intermediate-sized peptide hormones, many of which are implicated in skin health and wound healing. Pharmacological inhibitors of IDE administered internally have been shown to slow the breakdown of insulin and thereby potentiate insulin action. Given the importance of insulin and other IDE substrates for a variety of dermatological processes, pharmacological inhibitors of IDE suitable for topical applications would be expected to hold significant therapeutic and cosmetic potential.

Temporal Labeling of Nascent RNA Using Photoclick Chemistry in Live Cells.

We report the first cellular application of a photoclick SPAAC reagent to label azide-functionalized RNA. 350 nm irradiation of a cyclopropenone caged oxo-dibenzocyclooctyne (photo-ODIBO) biotin yields formation of the SPAAC reactive species, which rapidly forms adducts with RNA containing 2'-azidoadenosine (2'N-A). Photo-ODIBO was found to be highly stable in the presence of thiols, conferring greater stability relative to ODIBO.

Cell-Selective Bioorthogonal Metabolic Labeling of RNA.

Stringent chemical methods to profile RNA expression within discrete cellular populations remains a key challenge in biology. To address this issue, we developed a chemical-genetic strategy for metabolic labeling of RNA. Cell-specific labeling of RNA can be profiled and imaged using bioorthogonal chemistry.

Evolving insights into RNA modifications and their functional diversity in the brain.

In this Perspective, we expand the notion of temporal regulation of RNA in the brain and propose that the qualitative nature of RNA and its metabolism, together with RNA abundance, are essential for the molecular mechanisms underlying experience-dependent plasticity. We discuss emerging concepts in the newly burgeoning field of epitranscriptomics, which are predicted to be heavily involved in cognitive function. These include activity-induced RNA modifications, RNA editing, dynamic changes in the secondary structure of RNA, and RNA localization.

Metabolic Incorporation of Azide Functionality into Cellular RNA.

Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2'- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA.

Chemical Tools for Dissecting the Role of lncRNAs in Epigenetic Regulation.

Proper control and maintenance of gene expression is critical for cellular identity and maintenance. Transcription of RNA from the genome is intimately controlled by post-translational chemical modification of histone tails and DNA. Recent studies have demonstrated that chromatin-remodeling complexes seek out their target genomic loci through the help of noncoding RNA molecules.