Super-resolution imaging of a 2.5 kb non-repetitive DNA <i>in situ</i> in the nuclear genome using molecular beacon probes.

Abstract

High-resolution visualization of short non-repetitive DNA <i>in situ</i> in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence <i>in situ</i> hybridization (FISH) using Oligopaint probes have enabled super-resolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used three-dimensional stochastic optical reconstruction microscopy (3D-STORM) with optimized imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and only 29-34 individual MB probes, we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA <i>in situ</i> in human or mouse genome, respectively. We demonstrated our MB-based FISH method was capable of visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution.