Single molecule mass photometry of DNA and DNA-protein interactions

Thesis

Mass photometry (MP) based on interferometric scattering microscopy (iSCAT) is a novel, label-free imaging and quantification approach, which allows for the accurate mass measurement of biomolecules as well as the quantification of protein-protein interactions in solution. In this work, we show that this approach is equally applicable to DNA and DNA-protein interactions.

After illustrating iSCAT's ability to visualise the crystallisation events of non-biological systems, including alkali-metal halides and metal-organic frameworks, we switch to demonstrating the capability of MP to directly measure relative concentrations by molecular counting in complex DNA mixtures. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy.

We then present an experimental assay to measure the key interactions responsible for the cohesin assembly, its interactions with DNA, and their modulations by ATPase cycle, providing new insights into studying complex biomolecular assemblies and interactions with the multimeric nature.

Lastly, we introduce surface passivation to control the binding density of biomolecules to cope with increasing measurement complexity, introducing a path to detect low-abundance biomolecules of interest without a pre-purification step, also with the potential to measure low-affinity interactions.

These results together highlight mass photometry as a label-free, rapid, and accurate single molecule method complementary to existing techniques for characterising and quantifying DNA and DNA-protein interactions.

Authors: Li, Y

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: loop extrusion; crystallisation of MOFs; surface passivation; DNA-protein interactions; mass photometry; cohesin; interferometric scattering microscopy; single molecule detection and quantification
• Subjects: DNA-binding proteins; Nucleic acids--Analysis; Imaging systems in biology; DNA-protein interactions