APEX-seq, an acronym for Activity-based Protein Enrichment followed by Sequencing, enables scientists to precisely map the location of proteins and RNA molecules within living cells. By combining a unique enzyme with sequencing technologies, APEX-seq offers a detailed look into the cellular environment. It provides a means to explore the intricate spatial organization of molecules, which is fundamental to understanding how cells function.
What APEX-seq Reveals
APEX-seq provides a high-resolution view of the cellular landscape. It maps the proximity of proteins and RNA molecules within living cells, identifying those within approximately 20-25 nanometers of a selected target. This spatial information is valuable for understanding how cellular components are organized and interact within specific compartments.
The output from APEX-seq experiments generates a “spatial map” of the transcriptome, the complete set of RNA molecules in a cell. For instance, studies have used APEX-seq to map RNA localization across nine distinct subcellular regions in human cells, including the nucleus, mitochondrial membrane, and endoplasmic reticulum. This mapping shows how different RNA classes and their variations are distributed throughout the cell.
The Core Mechanism of APEX-seq
APEX-seq relies on an engineered enzyme called APEX2, a modified ascorbate peroxidase. This enzyme is fused to a protein or peptide, directing it to a specific cellular location like the outer mitochondrial membrane or nucleus. Once localized, APEX2 is activated by introducing biotin-phenol and hydrogen peroxide into the living cells.
Upon activation, APEX2 generates highly reactive, short-lived free radicals from the biotin-phenol substrate. These radicals quickly react with and covalently attach biotin tags to nearby proteins and RNA molecules within approximately 20-25 nanometers. This labeling occurs rapidly, within one minute, ensuring only molecules in close proximity to APEX2 are tagged. The reaction is then stopped, and biotinylated molecules are isolated using streptavidin beads, which have a strong affinity for biotin. The tagged RNA molecules are subsequently sequenced to identify them and determine their precise localization.
Real-World Applications of APEX-seq
APEX-seq has expanded our understanding across various biological processes by providing detailed spatial information about molecular interactions. It has been used to map the spatial organization of the transcriptome at nanometer resolution, showing how different RNA classes and their isoforms are distributed within human cells. This has uncovered insights into the radial organization of the nuclear transcriptome and how messenger RNA (mRNA) is exported from the nucleus.
The technique has also elucidated mechanisms of gene regulation by showing how RNA molecules localize to specific cellular regions, influencing protein production. For example, APEX-seq has identified two distinct pathways for mRNA localization to mitochondria, each linked to specific sets of transcripts involved in building mitochondrial machinery. APEX-seq contributes to understanding disease mechanisms, such as in neurodegenerative diseases, by mapping the RNA composition of dynamic structures like stress granules. It helps reveal how the organization of translation initiation complexes on active mRNAs affects cellular function.
APEX-seq in the Molecular Biology Toolbox
APEX-seq offers unique advantages within molecular biology. Unlike methods requiring cell fixation or extensive biochemical fractionation, APEX-seq performs proximity labeling directly in living cells, preserving the native cellular environment. This live-cell labeling, completed within about one minute, provides high temporal and spatial resolution, capturing molecular interactions missed by slower methods.
The technique surveys the local proteome and transcriptome, allowing a comprehensive view of both protein and RNA components in specific subcellular compartments. It can effectively map the RNA content of regions previously difficult to access with other techniques. APEX-seq can also detect various RNA types, including long noncoding RNAs (lncRNAs) and antisense RNAs, which are often not captured by methods focused solely on actively translating mRNAs. This versatility makes APEX-seq well-suited for investigating the dynamic behavior of RNA and protein-RNA interactions at length scales beyond direct physical contact but too short for traditional microscopy.