Cycloaddition enabled mutational profiling of 5-vinyluridine in RNA.

We report the detection of 5-vinyluridine (5-VUrd) in RNA at single nucleotide resolution via mutational profiling. Maleimide cycloadducts with 5-VUrd in RNA cause a stop in primer extension during reverse transcription, and the full-length cDNA product from reverse transcription contains misincorporation across the cycloadduct site.

Exploiting Endogenous Enzymes for Cancer-Cell Selective Metabolic Labeling of RNA in Vivo.

Tissues and organs are composed of many diverse cell types, making cell-specific gene expression profiling a major challenge. Herein we report that endogenous enzymes, unique to a cell of interest, can be utilized to enable cell-specific metabolic labeling of RNA. We demonstrate that appropriately designed "caged" nucleosides can be rendered active by serving as a substrate for cancer-cell specific enzymes to enable RNA metabolic labeling, only in cancer cells. We envision that the ease and high stringency of our approach will enable expression analysis of tumor cells in complex environments.

Electrostatic and Covalent Assemblies of Anionic Hydrogel-Coated Gold Nanoshells for Detection of Dry Eye Biomarkers in Human Tears.

Although dry eye is highly prevalent, many challenges exist in diagnosing the symptom and related diseases. For this reason, anionic hydrogel-coated gold nanoshells (AuNSs) were used in the development of a label-free biosensor for detection of high isoelectric point tear biomarkers associated with dry eye. A custom, aldehyde-functionalized oligo(ethylene glycol)acrylate (Al-OEGA) was included in the hydrogel coating to enhance protein recognition through the formation of dynamic covalent (DC) imine bonds with solvent-accessible lysine residues present on the surface of select tear proteins. Our results demonstrated that hydrogel-coated AuNSs, composed of monomers that form ionic and DC bonds with select tear proteins, greatly enhance protein recognition due to changes in the maximum localized surface plasmon resonance wavelength exhibited by AuNSs in noncompetitive and competitive environments. Validation of the developed biosensor in commercially available pooled human tears revealed the potential for clinical translation to establish a method for dry eye diagnosis.

Chemical methods for measuring RNA expression with metabolic labeling.

Tracking the expression of RNA in a cell-specific manner is a major challenge in basic and disease research. Herein we outline the current state of employing chemical approaches for cell-specific RNA expression studies. We define the utility of metabolic labels for tracking RNA synthesis, the approaches for characterizing metabolic incorporation and enrichment of labeled RNAs, and finally outline how these approaches have been used to study biological systems by providing mechanistic insights into transcriptional dynamics. Further efforts on this front will be the continued development of novel chemical handles for RNA enrichment and profiling as well as innovative approaches to control cell-specific incorporation of chemically modified metabolic probes. These advancements in RNA metabolic labeling techniques permit sensitive detection of RNA expression dynamics within relatively small subsets of cells in living tissues and organisms that are critical to performing complex developmental and pathological processes. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Evolution and Genomics > Ribonomics RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry.

Nanogel receptors for high isoelectric point protein detection: influence of electrostatic and covalent polymer-protein interactions.

An aldehyde acrylate-based functional monomer was incorporated into poly(N-isopropylacrylamide-co-methacrylic acid) nanogels for use as protein receptors. The aldehyde component forms dynamic imines with surface exposed lysine residues, while carboxylic acid/carboxylate moieties form electrostatic interactions with high isoelectric point proteins. Together, these interactions effect protein adsorption and recognition.

Sequencing of Sequence-Defined Oligourethanes via Controlled Self-Immolation.

Sequence-defined polymers show promise for biomimetics, self-assembly, catalysis, and information storage, wherein the primary structure begets complex chemical processes. Here we report the solution-phase and the high-yielding solid-phase syntheses of discrete oligourethanes and methods for their self-immolative sequencing, resulting in rapid and robust characterization of this class of oligomers and polymers, without the use of MS/MS. Crucial to the sequencing is the inherent reactivity of the terminal alcohol to "unzip" the oligomers, in a controlled and iterative fashion, releasing each monomer as a 2-oxazolidinone. By monitoring the self-immolation reaction via LC/MS, an applied algorithm rapidly produces the sequence of the oligourethane. Not only does this process provide characterization of structurally complex molecules, it works as a reader of molecular information.