A new super-resolution proximity assay to probe RNA transcription condensates

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Although each cell has the same genetic information in the cell nucleus in the form of genomic DNA, which parts of this information are used ("transcribed") in a given cell to make proteins is closely regulated, allowing cells to fulfil a large number of different functions. The mechanisms that underlie the regulation of this transcription are incompletely understood despite their fundamental importance for every organism. Recently it has been discovered that there are specialised microscopic regions within the cell nucleus where molecules involved in genetic transcription are concentrated. To establish the roles of these microscopic regions or "condensates" we will study which molecules interact in them and how these interactions change when the cell is exposed to stress.Detecting and quantifying interactions within the microscopic condensates is difficult using traditional methods due to their multiplicity and very small size. We will therefore develop and apply a new microscopy technology to dissect the molecular interactions with very high spatial resolution so that we can directly see (i) which molecules interact, (ii) where these molecules interact and (iii) how many molecules interact in this way, all within the microscopic condensates. To achieve these stringent requirements we will use methods of nanotechnology that employ synthetic DNA sequences to test if two molecules are close to each other (say within 10 nm) and image this with the very high resolution provided by a range of microscopy techniques termed "optical super-resolution microscopy" which allow detection and localisation of such protein pairs to within ~ 10 nm.This project will focus on adapting and enhancing the new technology, termed ePD-PAINT (enhanced proximity-dependent PAINT), by using the principles of DNA nanotechnology and super-resolution imaging. ePD-PAINT will be first validated with synthetic test samples, made of DNA itself, and termed "DNA origami" in analogy with the art of paper folding, folding DNA molecules to defined molecular patterns. The improved ePD-PAINT approach will then be validated in biological cells using assays where proteins can be manipulated to induce molecular interactions in response to an analogue of the small chemical rapamycin, which is also used as an immune-suppressant. To complete validation, the measurements with the ePD-PAINT technology will be compared to classical biochemical protein interaction assays.We will investigate the interactions between several key proteins involved in transcription using ePD-PAINT directly in the microscopic condensates and test how these interactions change when transcription is impaired, either by interventions in the steps performed during transcription or by exposing the cell to external stress which is known to change transcription as the cell responds and adapts to the external constraints.These findings will have significant impact on basic cell biology and enable a deeper understanding of protein-protein interactions in regions where the gene transcription machinery is concentrated. Furthermore, they may come to underpin a major new mechanism underlying the regulation of gene expression. Not only is this important for all of cell biology, but it may also inform novel strategies to manipulate the process when errors occur during disease. Of note, many of the proteins known to accumulate in condensates (including BRD4 as will be studied here) are implicated in cancer and neurodegeneration.