Abstract
DNA single-molecule fluorescence in situ hybridization (smFISH) is a powerful technique used to visualize intracellular DNA. However, the technique suffers from a fundamental limitation in the annealing (or hybridization) efficiency of the labeled probes. This is largely due to competition from the complementary DNA strand, which can similarly anneal to the target. With the invention of artificial DNA mimics such as peptide nucleic acids (PNAs), which contain a neutral backbone, the hybridization efficiency and specificity of smFISH can be significantly increased due to the reduced electrostatic repulsion between the PNA probes and DNA target. Using super-resolved localization microscopy with PNA smFISH, we are investigating the spatial organization and maintenance of high-copy number plasmids in bacteria. In the future, this technique may also be fruitful in imaging genetic loci within the chromosome and, because PNA can strand invade dsDNA, live-cell imaging.
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