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

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.

Recently it has been discovered that RNA polymerase II (Pol II) and many transcription factors are concentrated in microscopic regions, or 'condensates', that result from phase separation due to disordered protein domains that promote aggregation. The condensates are microscopic in size (<500 nm) and have only recently been characterised. We currently lack methods to probe the molecular interactions within these condensates and pinpoint where within the microscopic condensate these interactions occur (e.g. at the periphery, in the center, etc). To enable dissection of these interactions which hold the key to the function of condensates in transcription, we will develop a novel super-resolution imaging method that is sensitive to the proximity of two molecules. The imaging approach, termed 'proximity-dependent PAINT' (PD-PAINT), exploits principles of the super-resolution method DNA-PAINT with which it shares the use of synthetic DNA strands. We will provide an enhanced mode that we call ePD-PAINT by maximising efficiency of pair detection. We will first validate ePD-PAINT with synthetic nanotemplates which allow us to place molecules at well-defined distances from each other. Validation in cells will use FKBP and FRB containing constructs as chemically inducible interaction pairs, using rapamycin analogues to induce dimerisation. The validated ePD-PAINT assay will be applied to dissect molecular interactions in Pol II, Mediator and BRD4-containing condensates, and how these change throughout the transcription cycle or when the cell is exposed to stresses that cause large scale re-wiring of gene expression. Our findings will inform the mechanistic thinking on how transcription is regulated in human cells, which underpins all biological processes. There are numerous instances where the ability of the novel ePD-PAINT proximity assay will transform our understanding of cell biology and we expect the technology to be widely adopted for these purposes.

A broad range of groups and stakeholders will benefit from the proposed project. These include:

- Biological scientists and other academic beneficiaries
- General imaging and biotechnology manufacturers
- The pharmaceutical and wider biotechnology industry

Ways in which these groups will benefit
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The developments that we will pursue will allow commercial manufacturers to increase the capabilities of their microscopy and labelling technologies and offer improved and new products that should create demand in their respective markets.

We anticipate increasing use of super-resolution methods in biological research and applications, so that technologies aiming at improving the capabilities of super-resolution imaging approaches will be in high demand. It is critical to demonstrate the applicability and versatility of the enhanced proximity methods which we will achieve by evaluating our technology in multiple cellular compartments and by dissecting the molecular make-up and dynamics of Pol II condensates in the cell nucleus.

The results of this project will be of interest to the microscopy industry in the field of super-resolution. The PI has a strong track record of collaborative working with this industry. To realise the impact the researcher team has partnered with Bio-Techne as a project partner who will provide commercialisation support. To maximise effective impact engagement with stakeholders we will also conduct a workshop with industry and academic participants. Commercialisation of the IP arising from this project will be pursued in continuation of the efforts to commercialise PD-PAINT technology for which a patent application has already been filed.

The types of benefit outlined above make a contribution to the nation's health and wealth and help to enhance quality of life and long term health as insight from these applications ultimately may inform improved health care.

The research and technical staff working on this project will acquire advanced skills in cell biology, super-resolution imaging, DNA nanotechnology, and related advanced biotechnology relevant approaches. The PDRAs in particular will acquire and refine a set of skills that should make them sought after for employment in biotechnology, pharmaceutical, advanced application development and a variety of research and development roles.

We will also engage with the general public via outreach events where we will showcase both microscopy activities and the biology of gene regulation as the public has an increasing interest in how genes influence the fate of organisms, and that of us humans in particular.