Optimisation of DNA-PAINT super-resolution imaging of proteins in the nucleus of mammalian cells and application to RNA polymerase II

Abstract

The DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful and increasingly adopted method in the field of super-resolution microscopy. It attracts the user for its ease of implementation, lack of complex fluorophore photophysics requirement to induce blinking, as well as a virtually unlimited pool of fluorophores thereby offering an extensive photon budget. However, the DNA itself, which is an integral part of the technique, has a tendency for non-specific accumulation in the nucleus creating a substantial background signal that prevents from visualising intranuclear molecules. Given its pivotal role in various cellular regulatory processes, such as DNA transcription, and housing an abundance of key biomolecules involved in these processes, the nucleus is an unquestionable area of interest for advanced nanoscale imaging. While the role of imagers in generating a background signal has been previously recognised and addressed, this thesis sheds light on an additional and even more significant contributor or source of the issue which are the docking strands. A few strategies have been developed to overcome this limitation. Firstly, it has been found that the non-specific nuclear staining can be reduced to some extent via an increase of sodium chloride in the secondary antibody incubation buffer. Even though the efficacy of this solution varied and was observed to be sequence-dependent, it remains a valuable approach owing to its simplicity and cost-effectiveness. Further progress has been made through the development of novel docking strand designs and a hemi-duplex system. Such modification of the docking strands is ultimately proposed as a robust and universal approach that minimises the problem of non-specific signal in the nucleus. Additionally, this work highlights that optimising the procedures of sample preparation is a critical aspect that has significant implications for the overall success and validity of the research, especially in super-resolution microscopy techniques like DNA-PAINT. Furthermore, the optimised procedures and the proposed technology that mitigates the non-specific background issue is applied on a true intranuclear target RNA polymerase II. The successful adaptation of DNA-PAINT for nuclear targets serves as an important milestone in the field which ultimately opens new avenues for further, more in-depth studies of gene regulation, transcription, and associated processes at the nanoscale.