ICMCS Seminar - Separating DNA Topologies using Fluorescence Microscopy

DNA is ingeniously packed into complex and compact superstructures called chromosomes. For vital cellular processes such as replication (cell division) and transcription (protein synthesis) to occur, DNA must be unwound, exposing the information-rich base sequence.

Accessing genetic information from DNA imparts topological stresses, manifesting as supercoils (over/undertwisted DNA) and catenanes (interlinked DNA) which become lethal if unresolved. Topoisomerases are homeostatic enzymes which relieve this stress and are overexpressed in rapidly dividing cancer cells, making them key targets for chemotherapy. Gel electrophoresis is crucial to separating and quantifying DNA fragments based on their size and topology. When an electric field is applied, charged DNA molecules travel through a gel matrix. More compact, supercoiled topologies travel farther than relaxed, unstressed DNA. Similarly, unlinked DNA travels farther than their larger, catenated counterparts. These distinct topologies are clearly resolved using gels. However, the process is time-consuming, often requiring several hours to days to run. We show DNA topologies can also be differentiated using fluorescence microscopy. Labelled DNA fluoresces as it moves through solution and from extracting its diffusive motion, we can size the DNA. Importantly, we show this is true when the DNA is vastly beyond the resolution limit of the microscope. This sample preparation and analysis can be completed within 5 minutes per sample, offering a viable, novel technique to perform biophysical assays with superior efficiency than gel electrophoresis. We present imaging results for large catenated DNA(3-5 µm) to smaller catenanes(180 nm) and additionally show that microscopy can resolve decatenated intermediates.

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