Targeting Twist: Single-molecule insights into supercoiled DNA-topoisomerase interactions for drug discovery

Lead Research Organisation: University of Sheffield
Department Name: Materials Science and Engineering

Abstract

Topoisomerases are enzymes, present in all organisms from humans to bacteria, which are essential for life due to their ability to untangle knotted and twisted DNA. The knotting and twisting of our DNA occurs as 2 metres of our DNA is folded into the cell nucleus; much narrower than the width of a human hair. This is exacerbated by the molecular machinery in our cells which travel along our DNA, pulling it apart and manipulating it, in a compact environment. Without topoisomerases, our DNA becomes irreversibly knotted and twisted, and the cell will die. For this reason, key therapeutics such as anticancer and antibiotic drugs target human and bacterial topoisomerases respectively either killing cancerous or bacterial cells.

The pharmaceutical pipeline is continually working both to improve the therapeutics that we have, and to create novel therapeutics. To improve existing therapeutics, we require better understanding of how they work. There remains much room for development in this field. Anti-cancer therapeutics that target topoisomerases currently suffer from a lack of selectivity. Further to this, there remain a number of unexplored antibacterial targets, which have significant potential, but are as yet untargeted.

I propose to use single molecule techniques such as Atomic Force Microscopy, where a needle moves up and down as it is scanned over a surface, building up a picture of what the surface looks like, line by line, (much like a record player reads out a record by feeling the contours of its surface). These will be complemented by faster single molecule techniques, such as magnetic tweezers where a piece of DNA is tethered to a magnetic ball at one end, and a glass surface at the other. The DNA is twisted into a more complex state, by rotating the magnetic ball. The length of DNA is determined by measuring the height of the ball. This gives a very fast readout of how topoisomerases are able to untwist DNA, and how this process can be disrupted by drugs such as anti-cancer therapeutics and antibiotics. The aim of the project is to improve our understanding of how topoisomerases untwist DNA, and how this is prevented by topoisomerase inhibitors, to aid in the development of new or improved anti-cancer drugs and antibiotics.

Technical Summary

Topoisomerases are molecular machines that perform vital rearrangement of DNA, unknotting and decatenating it in order that transcription machinery can pass along genomic DNA unhindered. Topoisomerases are targeted by antibiotic and anti-cancer therapeutics that trap them on DNA, reducing their activity. This mechanism of action, discovered for topoisomerase inhibitors, also extends to PARP inhibitors used in anti-cancer therapies, and may be applied to target transcription factors. There remains much room for development in this field; anti-cancer therapeutics that target topoisomerases currently suffer from a lack of selectivity, and there remain a number of unexplored antibacterial targets (including topoisomerases I and III), which have significant potential, but are as yet untargeted. This is due to both a lack of structural information and fundamental gaps in our knowledge of their mechanism of action. Using single molecule techniques, I will provide valuable insight into the mechanism of action for topoisomerases and their inhibitors, with the aim of informing the rational design of novel therapeutics.

This work will bring together an international multidisciplinary collaboration. Using Atomic Force Microscopy techniques I have pioneered, I will determine the conformation of DNA molecules, as they interact with topoisomerases, at double-helical resolution in aqueous solution. Using magnetic tweezers and single molecule fluorescence, I will interrogate topoisomerase-DNA interactions at millisecond time scales, providing dynamic insights into the binding of these enzymes, and how this is modulated by novel therapeutics such as anticancer drugs and antibiotics. These pioneering studies will allow for the determination of the supercoiling dependence of topoisomerases, and provide insight into the mechanism of topoisomerase-DNA interactions, and how these are modulated by topoisomerase inhibitors, a key class of therapeutics.
 
Description DTP 2018-19 University College London
Amount £15,585,724 (GBP)
Funding ID EP/R513143/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 08/2018 
End 09/2023
 
Description EPSRC DTP studentship deep probabilistic models for analysing complex DNA structures in high-resolution atomic force microscopy images
Amount £120,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 09/2021 
End 05/2025
 
Description EPSRC DTP studentship: Tracking and tracing complex DNA structures critical to human health
Amount £80,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 09/2021 
End 04/2025
 
Description Henry Royce Industry Collaboration Partnership
Amount £37,319 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 04/2021 
End 10/2021
 
Description Near-Field Optical Spectroscopy Centre at Sheffield, NOSC
Amount £1,656,502 (GBP)
Funding ID EP/V007696/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 11/2020 
End 11/2024
 
Description Sir Henry Royce Institute - recurrent grant
Amount £31,477,552 (GBP)
Funding ID EP/R00661X/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 03/2016 
End 03/2023
 
Title TopoStats 
Description Atomic Force Microscopy (AFM) can image single molecules on surfaces with Ångström resolution, without the need for labelling or averaging. One of the rate-limiting steps for AFM is in the reproducibility of analysis of the increasing volumes of data produced. Even today the majority of AFM analysis has been carried out by hand, relying on a highly trained and experienced researcher. Unlike for other imaging modalities, there are few open-source analytical pipelines available and arguably none that have been adopted by the community. There is a need for automated analysis in AFM to reduce reliance on an experienced researcher, minimise selection bias and facilitate rapid quantitative analysis. We have developed an AFM image analysis platform TopoStats (https://github.com/AFM-SPM/TopoStats); a first of its kind Python toolkit for automated AFM data processing and analysis. This tool can identify individual biomolecules and provide quantitative insight into their size, and aggregation. 
Type Of Material Improvements to research infrastructure 
Year Produced 2021 
Provided To Others? Yes  
Impact This tool is bring used by researchers across the UK, via the GitHub platform, and is having impact across sectors 
URL http://www.github.com/AFM-SPM/TopoStats
 
Title Atomic force microscopy and atomistic molecular dynamics simulation data to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. 
Description This data record consists 2 zipped folders: Full AFM raw data set.zip, and Source data .zip.The zipped folder Full AFM raw data set.zip contains all raw AFM data including repeats and experiments carried out in alternative conditionsThe primary subfolder names correspond to the method of DNA immobilisation:Nickel - use of 3 mM NiCl2 in Ph7.4 20 mM HEPES bufferPLLNaOAc - use of PLL and pH 5.4 50 mM NaOAc bufferHR images - high resolution images, obtained also using the nickel conditions.The secondary subfolder names correspond to the superhelical density as shown in figure 3 in the article, and these contain the raw AFM images as .spm isles, the sub folders within those are created by the program TopoStats, and are processed data from the raw AFM images. File formats included in the zipped folder: .spm, .tiff, .json, .txt and .pdf.The zipped folder Source data .zip comprises all relevant data, pdbs of all the structures depicted in the paper obtained from simulations and AFM. See below for details on each sub folder within Source data 2.zip. Each folder contains the data used to generate each figure ad supplementary figure in the article. Figure 1: AFM data: the AFM raw files for the high-resolution images shown in figure 1, and calculations of their aspect ratios as aspectratiomanual.xlsxAFM movie: the AFM raw files for the time-lapse images shown in figure 1.MD data: the MD images used for the high-resolution images shown in figure 1 and .tar files - the MD files used to generate the snapshots MD movie: the MD snapshots files for the time-lapse images shown in figure 1 and .tar files - the MD files used to generate the snapshotsFile formats included in the Figure 1 sub folder: 0## files where ## represent numbers, .gwy, .txt, .eps, .mpg and .xlsx.Figure 2: Kink and defect measurements - the measured bend angles shown in Fig 2 and an AFM image showing how the FAM bends were measuredMD Radgyr Writhe - measurements of radius of gyrations and writhe for each topoisomer.tar files - the MD files used to generate the snapshots in 2a.txt file - the profile shown in fig 2bFile formats included in the Figure 2 sub folder: .tiff, .txt and .datFigure 3: The subfolder names correspond to the superhelical density as shown in figure 3, and these contain the raw AFM images as '.spm' isles, the sub folders within those are created by the program TopoStats, and are processed data from the raw AFM images. The '.json' file contains the data used to make the plots shown in Figure 3File formats included in the Figure 3 sub folder: .spm, .tiff, .txt, .json and .pdfFigure 4: '.dat' files contain information from MD simulations used to create the subfigure they are labelled with.The '.spm' and '.037' files are the raw AFM images used in this figure.The .tar files are MD simulations data used to generate the snapshots shown in figure 4.File formats included in the Figure 4 sub folder: .spm, .txt, .pdf and .datFigure S1: Simulations data generated using the SerraLine program, showing the average and maximum deviations from planarity in relative and absolute numbers.Data were plotted suing the distributions_plot.py script.File formats included in the Figure S1 sub folder: .csv, .pdf, and .txtFigure S2a: MD measurement of the writhe over time as a '.dat file' and snapshots as '.pdb' files. File formats included in the Figure S2a sub folder: .pdb and .dat.Figure S2b: MD measurement of the writhe over time as a '.dat file' and snapshots as '.pdb' files.File formats included in the Figure S2b sub folder: .pdb and .dat.Figure S3: The AFM and MD measurements of bending angles including all profiles for MD simulations, generated using Serraline A, FM images and measurements in the form '251angles' '339 angles'.File formats included in the Figure S3 sub folder: .tiff, .txt and .pdb.Figure S4: AFM length analysis of the position of the triplex on linearised minicircles. 'Csv' file contains the length data measured by hand using the IMOD software.Plots: plots of the data raw AFM data: AFM data files used in the analysisFile formats included in the Figure S4 sub folder: .csv, .xlsx, .pdf and 0## files where ## represent numbers.Figure S5: Surface plasmon resonance (SPR) data show the effect of ions on the affinity of the triplex for varying superhelical densities of DNA minicircles, plotted using the script 'sprplot'. '.pdf' files are the plots of the various excel files.File formats included in the Figure S5 sub folder: .json, .pdf and .xlsx.Figure S6: SPR data in showing the affinity of the triplex for varying superhelical densities of DNA minicircles, plotted using the script 'sprplot'. '.pdf' files are the plots of the various excel files.File formats included in the Figure S6 sub folder: .json, .pdf, .xlsx and .pdf.Figure S7: An MS '.tar' file containing the snapshots shown in figure S7File formats included in the Figure S7 sub folder: .pdbFigure S8: AFM data used in figure s8, the '.gwy' files are AFM images of the wide view, and each of the time-lapse images. The '.txt' files are the profiles taken in those images and plotted in the figure.File formats included in the Figure S8 sub folder: .gwy and .txt.Figure S9: Simulations data showing the difference between the OL$ and BSC1 forcefields.File formats included in the Figure S9 sub folder: .datSimulations: The simulations data File formats included in the Simulations sub folder: .gro and .xtcSupp videos: The supplementary videosFile formats included in the SuppVideods sub folder: .pdb and .mpgSoftware needed to access data: 20151103_251_NAT_17ng_Ni_20mm_052DX.058 or AFM_339_TFO_HR_cs.037, spm files & all files included in the "Raw AFM data" sub folder - Gwyddion, Nanoscope Analysiseps files - illustrator/ pdf software.mpg - any movie player.gro - gromacs files- GRO files may be viewed on a computer using a supporting HP calculator emulator, such as Emu48.xtc files - gromacs files- a suitable software like XTrkCADsee http://manual.gromacs.org/documentation/2018/user-guide/file-formats.html for more information on gromacs files.Study aims and methodology: In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. In this study, the authors combined high-resolution atomic force microscopy (AFM) with molecular dynamics (MD) simulations to reveal how supercoiling affects global and local DNA conformation, structure and dynamics in DNA minicircles of length 250-340 bp. The following procedures are described in more detail in the related article: generation and purification of small DNA circles, preparation and analysis of different topological species of minicircles, S1 nuclease digestions, atomic force microscopy, atomistic simulations and surface plasmon resonance. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact Dataset of full data published in Nature Communications 
URL https://springernature.figshare.com/articles/dataset/Atomic_force_microscopy_and_atomistic_molecular...
 
Title TopoStats 
Description We present TopoStats, a Python toolkit for automated editing and analysis of Atomic Force Microscopy images. The program includes identification and tracing of individual molecules in both circular and linear conformations without user input. The program is freely available via GitHub (https://github.com/afmstats/TopoStats), and is intended to be modified and adapted for use if required. TopoStats can identify individual molecules and molecular assemblies within a wide field of view, without the need for prior processing. We demonstrate its power by identifying and tracing individual biomolecules, including DNA origami, pore-forming proteins, and DNA molecules in both closed circular and linear form. 
Type Of Material Computer model/algorithm 
Year Produced 2021 
Provided To Others? Yes  
Impact Software published in Elsevier Methods, and used as a community resource for the data analysis in AFM field 
URL http://www.github.com/AFM-SPM/TopoStats
 
Description Bruker collaboration 
Organisation Bruker Corporation
Department Bruker Nano-surfaces Division
Country United States 
Sector Private 
PI Contribution Bruker nano surfaces develop Atomic Force Microscopes and accessories for biological imaging, among other applications. I have worked in partnership with Bruker to develop novel probes for high resolution imaging, and to optimise their software and imaging modes to obtain the highest resolution imaging. We work together to bring these together in the form of protocols for high resolution imaging of biomolecules.
Collaborator Contribution Bruker provide me with pre-release probes for testing as part of their development process. In addition they provide me with unrestricted access to their state of the art facilities in Santa Barbara, allowing me to use optimised machines for my research.
Impact Bruker have developed new probes and protocols for high resolution imaging of DNA as part of this collaboration which is now commercially available
Start Year 2015
 
Description DNA minicircle collaboration 
Organisation University of Leeds
Department School of Physics and Astronomy
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental/simulation partnership to determine the effect of supercoiling on DNA structure
Collaborator Contribution The John Innes centre made up DNA mini circle constructs and provided expertise in DNA supercoiling and recognition. They carried out SPR and other biochemical experiments and training for the PI in biochemical analysis. The University of York and Leeds carried out simulations to correlate to our experimental work, and developed analysis tools to allow us to compare our experiments explicitly to their simulations. The University of Liverpool provided expertise in DNA supercoiling
Impact Multidisciplinary Biophysics collaboration - the effect of DNA supercoiling on molecular recognition. Preprint Published paper - Nature Communications Seminars x4 Conference presentation x6 Software
Start Year 2017
 
Description DNA minicircle collaboration 
Organisation University of Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental/simulation partnership to determine the effect of supercoiling on DNA structure
Collaborator Contribution The John Innes centre made up DNA mini circle constructs and provided expertise in DNA supercoiling and recognition. They carried out SPR and other biochemical experiments and training for the PI in biochemical analysis. The University of York and Leeds carried out simulations to correlate to our experimental work, and developed analysis tools to allow us to compare our experiments explicitly to their simulations. The University of Liverpool provided expertise in DNA supercoiling
Impact Multidisciplinary Biophysics collaboration - the effect of DNA supercoiling on molecular recognition. Preprint Published paper - Nature Communications Seminars x4 Conference presentation x6 Software
Start Year 2017
 
Description DNA minicircle collaboration 
Organisation University of York
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental/simulation partnership to determine the effect of supercoiling on DNA structure
Collaborator Contribution The John Innes centre made up DNA mini circle constructs and provided expertise in DNA supercoiling and recognition. They carried out SPR and other biochemical experiments and training for the PI in biochemical analysis. The University of York and Leeds carried out simulations to correlate to our experimental work, and developed analysis tools to allow us to compare our experiments explicitly to their simulations. The University of Liverpool provided expertise in DNA supercoiling
Impact Multidisciplinary Biophysics collaboration - the effect of DNA supercoiling on molecular recognition. Preprint Published paper - Nature Communications Seminars x4 Conference presentation x6 Software
Start Year 2017
 
Description John Innes Collaboration 
Organisation John Innes Centre
Department The Sainsbury Laboratory
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution As part of this partnership, we perform high resolution imaging of DNA -protein interaction, which are of interest to the group of Prof Tony Maxwell at the JIC
Collaborator Contribution Prof Maxwell's group provide us with protein and access to their wet lab facilities to run standard biochemical assays to complement our novel AFM analysis techniques.
Impact Maanuscript in preparation
Start Year 2015
 
Description TopoStats software development collaboration 
Organisation Birkbeck, University of London
Department School of Crystallography
Country United Kingdom 
Sector Academic/University 
PI Contribution We carried out experimental work and designed software to allow for quantitative analysis of atomic force microscope images
Collaborator Contribution Partners at Birkbeck developed more sophisticated algorithms for DNA tracing and tracking in Python which were included within our software.
Impact New software - TopoStats Published paper Preprint
Start Year 2019
 
Description TopoStats software development collaboration 
Organisation Birkbeck, University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution We carried out experimental work and designed software to allow for quantitative analysis of atomic force microscope images
Collaborator Contribution Partners at Birkbeck developed more sophisticated algorithms for DNA tracing and tracking in Python which were included within our software.
Impact New software - TopoStats Published paper Preprint
Start Year 2019
 
Title ANTI-MICROBIAL COMPOSITIONS COMPRISING NUCLEIC ACID NANOSTRUCTURES AND METHODS OF MAKING AND USING SUCH COMPOSITIONS 
Description Antimicrobial compounds are provided comprising: (i) a nucleic acid nanostructure; and (ii) a plurality of lipid molecules; wherein the plurality of lipid molecules are linked to the nucleic acid nanostructure. Compositions are provided with antimicrobial and antibiotic activity that comprise the described compounds. The compositions and compounds are selectively biocidal to microbes, especially bacteria, and not to eukaryotic cells. 
IP Reference WO2017118862 
Protection Patent application published
Year Protection Granted 2017
Licensed No
Impact Paper in preparation to show proof of principle work for DNA Nano-ores as antimicrobials
 
Title TopoStats - an automated tracing program for AFM images 
Description We present TopoStats, a Python toolkit for automated editing and analysis of Atomic Force Microscopy images. The program includes identification and tracing of individual molecules in both circular and linear conformations without user input. The program is freely available via GitHub (https://github.com/afmstats/TopoStats), and is intended to be modified and adapted for use if required. TopoStats can identify individual molecules and molecular assemblies within a wide field of view, without the need for prior processing. We demonstrate its power by identifying and tracing individual biomolecules, including DNA origami, pore-forming proteins, and DNA molecules in both closed circular and linear form. 
Type Of Technology Software 
Year Produced 2020 
Open Source License? Yes  
URL https://figshare.shef.ac.uk/articles/software/TopoStats_-_an_automated_tracing_program_for_AFM_image...
 
Description 3D Bionet invited talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Third sector organisations
Results and Impact 3D Bionet invited talk to discuss AFM as a bioimaging tool
Year(s) Of Engagement Activity 2019
 
Description DNA topology talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Third sector organisations
Results and Impact Talk and the DNA topology online series organised by Caroline Austin.
Year(s) Of Engagement Activity 2021
 
Description ITV interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Interviewed on ITV news about our Nature Communications paper https://www.nature.com/articles/s41467-021-21243-y
Year(s) Of Engagement Activity 2021
 
Description Interview for Henry Royce @ Sheffield 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Third sector organisations
Results and Impact This was a press release to document my joining Sheffield University as a lecturer in materials science and engineering and as head of the Royce nanocharacterisation laboratory
Year(s) Of Engagement Activity 2020
URL https://www.sheffield.ac.uk/materials/news/surface-characterisation-most-fundamental-level-atomic-fo...
 
Description MRC festival online 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact MRC online festical - attended online by hundreds of the general public. The research team made videos and other online activities for the public to engage with DNA supercoiling.
Year(s) Of Engagement Activity 2020
 
Description NuNano interview and blog 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact NuNano interview about open, interdisciplinary science
Year(s) Of Engagement Activity 2020
URL https://www.nunano.com/blog/2020/10/22/afm-community-interview-with-dr-alice-pyne
 
Description Royal Society fo Chemistry ECR meeting - invited speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Third sector organisations
Results and Impact Invited to give a talk on DNA supercoiling and how it affects molecular recognition as part of the RSC ECR meeting in Glasgow 2019
Year(s) Of Engagement Activity 2019
 
Description Seminar (St Andrews) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Third sector organisations
Results and Impact Seminar at St Andrews University to disseminate our recent work on DNA supercoiling https://www.biorxiv.org/content/10.1101/863423v1 - this sparked lively discussion and more traffic to website/papers
Year(s) Of Engagement Activity 2020
 
Description Seminar (Kent) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Third sector organisations
Results and Impact Seminar at Kent University to disseminate our recent work on DNA supercoiling https://www.biorxiv.org/content/10.1101/863423v1 - this sparked lively discussion and resulted in meetings with multiple academic groups in Kent
Year(s) Of Engagement Activity 2020
 
Description Seminar (Leeds) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Third sector organisations
Results and Impact Seminar at Leeds University in the biomaterials group to disseminate our recent work on DNA supercoiling https://www.biorxiv.org/content/10.1101/863423v1 - this sparked lively discussion and twitter contacts.
Year(s) Of Engagement Activity 2020