Cell-of-origin-specific proteomics of extracellular vesicles.

The ability to assign cellular origin to low-abundance secreted factors in extracellular vesicles (EVs) would greatly facilitate the analysis of paracrine-mediated signaling. Here, we report a method, named selective isolation of extracellular vesicles (SIEVE), which uses cell type-specific proteome labeling via stochastic orthogonal recoding of translation (SORT) to install bioorthogonal reactive groups into the proteins derived from the cells targeted for labeling. We establish the native purification of intact EVs from a target cell, via a bioorthogonal tetrazine ligation, leading to copurification of the largely unlabeled EV proteome from the same cell. SIEVE enables capture of EV proteins at levels comparable with those obtained by antibody-based methods, which capture all EVs regardless of cellular origin, and at levels 20× higher than direct capture of SORT-labeled proteins. Using proteomic analysis, we analyze nonlabeled cargo proteins of EVs and show that the enhanced sensitivity of SIEVE allows for unbiased and comprehensive analysis of EV proteins from subpopulations of cells as well as for cell-specific EV proteomics in complex coculture systems. SIEVE can be applied with high efficiency in a diverse range of existing model systems for cell-cell communication and has direct applications for cell-of-origin EV analysis and for protein biomarker discovery.

Chemoenzymatic strategies for RNA modification and labeling.

RNA is a central molecule in numerous cellular processes, including transcription, translation, and regulation of gene expression. To reveal the numerous facets of RNA function and metabolism in cells, labeling has become indispensable and enables the visualization, isolation, characterization, and even quantification of certain RNA species. In this review, we will cover chemoenzymatic approaches for covalent RNA labeling. These approaches rely on an enzymatic step to introduce an RNA modification before conjugation with a label for detection or isolation. We start with in vitro manipulation of RNA, sorted according to the enzymatic reaction exploited. Then, metabolic approaches for co- and post-transcriptional RNA labeling will be treated. We focus on recent advances in the field and highlight the most relevant applications for cellular imaging, RNA isolation and sequencing.

In Situ Hybridization for Detection of AAV-Mediated Gene Expression.

Techniques to localize vector transgenes in cells and tissues are essential in order to fully characterize gene therapy outcomes. In situ hybridization (ISH) uses synthesized complementary RNA or DNA nucleotide probes to localize and detect sequences of interest in fixed cells, tissue sections, or whole tissue mounts. Variations in techniques include adding labels to probes, such as fluorophores, which can allow for the simultaneous visualization of multiple targets. Here we provide the steps necessary to: (1) label probes for colorimetric visualization and (2) perform ISH on OCT cryo-preserved fixed frozen tissues.