A simple platform for in situ DNA-PAINT optimization enables improved multiplexed super-resolution imaging in Gram-negative bacteria
DNA-PAINT enables super-resolution imaging with high multiplexing capacity, yet it is rarely used in nanoscale bacteriology. A likely reason is the lack of straightforward protocols and of systematic characterization of imager-docking strand pairs inside intact bacterial cells, whose envelope and dense cytoplasm differ markedly from both the DNA origami in vitro systems used to develop them, and…
A streamlined platform for optimizing DNA-PAINT in situ facilitates enhanced multiplexed super-resolution imaging of Gram-negative bacteria. This innovation stems from the identification of obstacles hindering the widespread application of DNA-PAINT in nanoscale bacteriology. The primary challenge lies in the absence of user-friendly protocols and systematic evaluation of imager-docking strand pairs within the complex environments of bacterial cells.
To address this, researchers developed a screening platform employing RNA-FISH, which incorporated customizable docking sequences onto the 16S rRNA-directed EUB338 probe. This approach enabled rigorous assessment of imager-docking pairs directly inside living Escherichia coli cells, with a focus on critical parameters like specificity, binding kinetics, and localization precision.
Through extensive screening of 12 DNA-PAINT sequences, the team identified an optimized set for conducting four-target 3D Exchange-PAINT in individual E. coli cells. Furthermore, this strategy was successfully applied to visualize three effector mRNAs within the Gram-negative pathogen Yersinia enterocolitica, yielding detailed insights into the spatial arrangement and clustering of these molecules.
The presented framework and practical guidelines for high-quality 3D DNA-PAINT imaging in Gram-negative bacteria are expected to catalyze the broader adoption of this technique in nanoscale bacteriology research.
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