Babula, Anna Maria; (2024) Characterisation of the DNA trafficking mechanisms to improve nucleic acid therapeutics. Doctoral thesis (Ph.D), UCL (University College London).

Text UCL_PhD_Thesis_Final_Anna_Babula.pdf - Accepted Version Access restricted to UCL open access staff until 1 January 2026. Download (56MB)

Abstract

While nuclear accumulation of exogenous DNA in dividing cells is facilitated by the nuclear envelope breakdown, the biggest hurdle remains the delivery of DNA across the intact nuclear barrier. The efficiency of DNA nuclear import is low due to the large molecular weight of the encoded genes, which means that DNA’s size is above the passive diffusion limit. Additionally, the efficiency of this process is reduced because of a general lack of cellular specificity to accumulate exogenous DNA inside the nucleus. The majority of the cell targets for therapeutic application of DNA are non-dividing cells. Thus, exogenous DNA requires some manipulations on the sequence level to enhance its nuclear delivery through the nuclear barrier. Research regarding DNA nuclear import concludes that DNA (> 40 kDa) cannot enter the nucleus alone but it must interact with the nuclear proteins which mediate DNA’s nuclear delivery by interacting with the nuclear import receptors themselves. Thus, Touchlight Genetics have engineered DNA called doggybone DNA™ (dbDNA™) to accommodate unique DNA sequences that can elicit interaction with multiple nuclear proteins therefore, making this type of DNA more preferential for nuclear import, which is particularly essential in non-dividing cells. This project focused on designing a robust time-lapse microscopy assay that can serve as a tool to characterise new generations of dbDNA™ nuclear import rates with improved efficacy in non-dividing cells. The assay development included molecular biology methods in order to produce fluorescently labelled DNA, whose sequence encoded a fluorescent reporter spectrally well - separated from the labelling fluorophore. Three channels were used, phase contrast to capture the whole cell population in the field of view, rhodamine to capture labelled DNA and iRFP to capture reporter expression. A segmentation algorithm was trained on phase contrast images with annotated cell bodies or nuclei to infer single cells, regardless of the reporter expression. DNA distribution on a single-cell level, whether cytoplasmic or nuclear was measured by utilising the cellular compartments masks, while the reporter expression was quantified using the cell body masks. Although reporter expression analysis revealed differences between different types of dbDNA™ used, the observations in DNA signal tracking were not pronounced. An interesting observation specific to the DNA tested was the emergence of aggregates, which tended to increase in size, particularly in the cytoplasm. Further studies on this fascinating behaviour of DNA within the cellular environment remain a future objective to better understand the DNA cellular trafficking and as an end goal to increase the efficacy of DNA nuclear import.

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