Journal: Cell Reports Methods

Article Title: APEX2 proximity labeling of RNA in bacteria

doi: 10.1016/j.crmeth.2025.101206

Figure Lengend Snippet: APEX2 proximity labeling of RNA in bacterial cells with minimal perturbation (A) Schematic of APEX2 labeling of RNA protocol. The APEX2 protein was fused to RNase E, the major protein that scaffolds BR-bodies. Cells were incubated in medium containing alkyne-phenol, and labeling was initiated with H 2 O 2 . After a brief labeling reaction, RNA was extracted from the cells, and Cy5-azide was conjugated to the RNA by copper-catalyzed click chemistry. (B) APEX2 fusion does not dramatically impact localization or function (growth rate). See also and . Left: in vivo localization of RNase E-msfGFP vs. RNase E APEX2-msfGFP shows that fusion does not impact the formation of BR-bodies. Scale bars, 1 μm. Middle: growth of RNase E-APEX2 fusion is similar to wild-type Caulobacter cells. Right: mRNA half-life measurements by RT-qPCR show that RNase E-APEX2 degrades mRNAs with a similar half-life to wild type. Data are the averages from three biological and technical replicates and error bars represent standard deviation. (C) APEX2 labeling requires H 2 O 2 and alkyne-phenol to label RNAs. RNA labeling reactions were placed on a nylon membrane to bind to the RNA in a dot blot apparatus and scanned for Cy5 fluorescence in a gel imager. As a control, the RNA samples were incubated for 2 h with DNase I and RNase A. The RNA was then precipitated before being subjected to the azide-Cy5 click chemistry reaction and was re-precipitated before being filtered on the nylon membrane in the dot blot apparatus.

Article Snippet: The APEX2 gene can be obtained from Addgene (Cat# 129640).

Techniques: Labeling, Incubation, In Vivo, Quantitative RT-PCR, Standard Deviation, Membrane, Dot Blot, Fluorescence, Control

Journal: Cell Reports Methods

Article Title: APEX2 proximity labeling of RNA in bacteria

doi: 10.1016/j.crmeth.2025.101206

Figure Lengend Snippet: APEX2 proximity labeling of RNA works rapidly Optimization of APEX2 labeling of RNA with alkyne-phenol. Top: alkyne-phenol titration reveals that peak labeling occurs with 2.5 mM alkyne-phenol. RNA was labeled in the scheme shown in A, and the Cy5 intensity was measured in a gel imager. Bottom: time course of APEX2 labeling. RNA was labeled in the scheme shown in A, and RNA labeling is apparent in as little as 15 s of H 2 O 2 incubation, while peak labeling efficiency is observed at 45 s of H 2 O 2 incubation. RNA blots were obtained from two biological replicates. See also .

Article Snippet: The APEX2 gene can be obtained from Addgene (Cat# 129640).

Techniques: Labeling, Titration, Incubation

Journal: Cell Reports Methods

Article Title: APEX2 proximity labeling of RNA in bacteria

doi: 10.1016/j.crmeth.2025.101206

Figure Lengend Snippet: APEX2 proximity labeling of RNA works across species APEX2 requires heme for activity, so APEX2 fusions were generated in two additional species with heme biosynthesis pathways, B. subtilis (gram-positive) and E. coli (gram-negative). Both species were subjected to RNA proximity labeling in mid-exponential phase of growth in Luria Broth (LB) medium at 37°C, pre-incubated with alkyne-phenol for 30 min, and proximity labeling was triggered by a 45-s incubation with H 2 O 2 . RNA labeling reactions were subjected to azide-Cy5 click chemistry reactions and 5 μg RNA placed on a nylon membrane to bind to the RNA in a dot blot apparatus and scanned for Cy5 fluorescence in a gel imager. At least three biological replicates were carried out for each species.

Article Snippet: The APEX2 gene can be obtained from Addgene (Cat# 129640).

Techniques: Labeling, Activity Assay, Generated, Incubation, Membrane, Dot Blot, Fluorescence

Journal: Cell Reports Methods

Article Title: APEX2 proximity labeling of RNA in bacteria

doi: 10.1016/j.crmeth.2025.101206

Figure Lengend Snippet: APEX2-proximity-labeled RNA can be isolated by streptavidin purification (A) Schematic of the conjugation of biotin-azide to clicked alkyne-phenol and the resulting streptavidin purification. (B) Streptavidin purification of biotinylated RNAs. Agilent TapeStation RNA profiles of the lysates and elution fractions of biotinylated-proximity-labeled RNAs. The bottom two bands are tRNAs, which are known to be highly depleted in BR-bodies from differential-centrifugation-based isolation of BR-bodies. , (C) RNA-seq enrichment analysis of proximity-labeled RNA from BR-body + (RNaseE-APEX2, JS767) and BR-body− (RNaseEΔCTD-APEX2, JS803) cells. Log 2 ratio of elution/lysate from each sample is plotted for each RNA quantified. Data points were obtained from two biological replicates for each strain. Colored horizontal bars indicate the median values for each distribution. t test with uneven variance was used to compare the distributions of each RNA type, and RNA types with p < 0.05 are shown. See also .

Article Snippet: The APEX2 gene can be obtained from Addgene (Cat# 129640).

Techniques: Labeling, Isolation, Purification, Conjugation Assay, Centrifugation, RNA Sequencing