Single-molecule dynamics of human transcription regulation

Lead Research Organisation: University of Leicester
Department Name: Biochemistry

Abstract

My lab is interested in understanding how cells 'decide' what genes, and when, to switch on. Knowing how genes are regulated could, for example, provide clinicians with new drugs to cure genetic diseases, and scientists with new tools to turn regular cells into stem cells.

Previously, scientists had to infer how genes work from genetics (by following inherited traits), or from biochemical experiments (by grinding up cells and analyzing their chemical composition). However, no one has ever 'seen' a human gene molecule switch on, which limited our understanding of gene regulation. Recently, our lab has developed an imaging technology to directly 'see' gene molecules being switched on by single enzyme molecules. In this proposal, we will use our technology to reconstruct a minimal circuit of gene activation at single-molecule resolution, which will give us fundamentally new ideas on how genes are regulated.

Physically, genes are molecules of DNA located in the cell nucleus; most cells contain only two molecules of each gene. To switch a gene on means to make a protein based on the information encoded in the DNA. The cell decides to switch on a gene by recruiting an enzyme, called Pol II, to the gene molecule. Pol II then runs along the gene while making (transcribing) an 'active copy' of the gene, called RNA, which is then used as a template for making protein. The focus of this proposal is to find out how Pol II 'decides' to bind to a gene in the first place. This is an important question, because many genetic diseases (e.g. cancer) can be traced back to Pol II binding and copying a wrong set of genes.

Binding of Pol II to genes requires several other molecules, called transcription factors. Although most of transcription factors are known, the order in which they interact with each other to bring Pol II to a gene is not clear. By analogy, to understand football, it is not enough to identify each player in the field: one has to understand how the players interact with each other to get the ball (Pol II) between the goalposts (to a gene).

We propose to elucidate how transcription factors bring Pol II to a gene, literally, by watching the entire molecular game live. To do that, we will isolate a minimal team of players -- Pol II and five transcription factors -- from cells and mix them together in a drop of physiological solution. We will then put a single gene molecule on a microscope slide, cover it with the drop, and watch what is going on at the gene under a microscope in a dark room. To see the molecules of Pol II and transcription factors, we will label them with dyes of different colors (e.g. blue, green, and red), which will make the molecules glow against dark background like stars. By watching in what order the blue, green, and red stars bind to the gene, we will determine how the transcription factors bring Pol II to the gene.

After we elucidate how the minimal team of five factors plays the game, we will add one more player -- transcription 'activator' Sp1, which is present at high levels in rapidly dividing and cancerous cells -- and determine how Sp1 changes the game (e.g. with which transcription factors it interacts) to make Pol II produce more RNA copies of a gene.

Our analysis of how single molecules switch genes on may fundamentally change the way scientists think about gene regulation. In textbooks, switching a gene on is often shown as flipping on a switch. However, because each gene in a cell is represented by only two molecules, switching a gene on could be a stochastic ('sloppy') event, due to the Brownian motion of molecules in the microscopic world. Therefore, all decisions made by the cell (for instance, a decision to turn into a cancer cell) could be affected by stochastic collisions between Pol II, transcription factors and genes -- which may explain why some cell behaviors are difficult to control.

Technical Summary

Transcription of messenger RNA in the human cell begins with the assembly of RNA Polymerase II (Pol II) and General Transcription Factors (GTFs) on a promoter into a so-called preinitiation complex (PIC). Formation of the PIC is the step at which the cell commits to express a gene, and therefore is the main control panel for gene regulation. Although the key components of the PIC have been identified, the dynamics of PIC assembly, and the mechanism of PIC regulation are poorly understood.

We propose to apply a cutting-edge imaging technology to obtain a real-time, single-molecule 'movie' of PIC assembly and regulation. This new approach will reveal the full spectrum of dynamic interactions between Pol II and GTFs without introducing population-averaging effects, and will preserve the inherent stochastic, 'single-molecule' aspect of transcription in living cells.

The project has two specific objectives. In Objective 1, we will use a model cell-free system for 'basal transcription,' comprised of purified human Pol II and five GTFs (IIB, IID, IIF, IIE, and IIH) to determine in what order and at what rates GTFs and Pol II assemble on the promoter, which will pinpoint the rate-limiting steps of PIC formation. Furthermore, we will directly test whether GTFs remain at the promoter after Pol II starts elongation (a prototypical model for 'gene bookmarking').

In Objective 2, we will use a model cell-free system for regulated transcription, comprised of Pol II, GTFs, a prototypical human activator Sp1 and its co-coactivator IIA, to determine which step of PIC formation is targeted by activators to stimulate transcription, and how dynamic interactions between activator and co-activator molecules are integrated into the stimulatory signal.

This study will produce the first quantitative model of human transcription regulation, and will establish the benchmarks, technology and reagents for future studies of regulation of native genes in single living cells.

Planned Impact

This research project has a potential to have a great impact in the academy, industry, and in the wider public. We propose to build the first stochastic model of human transcription regulation based on quantitative data obtained using a cutting-edge single-molecule imaging technology.
1. Academic and industrial. This research will help understand how single cells make decisions to grow, differentiate or de-differentiate despite the stochastic noise of molecular collisions at promoters, and will establish fundamentally new ways of thinking about gene regulation. Thus, the immediate beneficiaries of this research will be researchers studying molecular mechanisms of gene regulation, and researchers using transcription factors as tools to control cell-state decisions. The insights into stochasticity of transcription will be of great interest to researchers studying dynamics of macromolecular complexes involved in nucleic acid transactions (e.g. translation, splicing and replication). Our protocols for fast, specific RNA hybridization will be of interest to researchers developing bottom-up bio-nanostructures. Our methods to generate bio-compatible surfaces have previously attracted, and will continue to attract the interest of companies working on high-throughput DNA sequencing and microfluidics. We will ensure that the academic and industrial beneficiaries are informed of our work by publishing in wide-audience peer-reviewed journals, attending scientific meetings in the UK and abroad, and keeping our lab web page up-to-date with the newest developments and publications. The cutting-edge research that we will bring to the University of Leicester will strengthen the position of the school as a hub for new technology, and attract young investigators from the USA and Europe who work at the interface of biology, chemistry, and physics.
2. Societal. The new insights into the mechanism of gene regulation that we will attain may provide researchers with better tools to control cell behavior, which may lead to better ways to treat diseases and engineer tissues, and, ultimately, improve the nation's health. The powerful visual aspect of our technology (using lasers to directly 'see' single enzyme molecules switching genes on) is very likely to attract the interest of the general public, in particular, middle- and high-school students. The students will be informed of our work through microscope-building and single-molecule-imaging demonstrations that we will organize with the GENIE Outreach program (see Pathways to Impact). The demonstrations will stimulate students' interest in biomedical sciences and nanotechnology, and, perhaps, encourage them to pursue careers in these high-tech fields -- which, indirectly, will have a long-term impact on the UK economy.
3. Career development. The postdoctoral research assistant and the research technician working on the project will learn a wide range of methods (single-molecule imaging and data analysis, molecular cloning, bioconjugation, surface chemistry, protein purification, instrument design, software development, cell culture, genome editing, and so on) and acquire critical thinking, presentation and writing skills which will prepare them for future careers in academia or industry, or for further education.
4. 'New ways of working'. This multidisciplinary study is within the scope of three strategic priorities of the BBSRC aimed at raising the awareness of novel, alternative methods of doing research: (i) technology development for the biosciences (bioimaging and functional analysis); (ii) data-driven biology (extracting quantitative information from large or complex image sets); and (iii) systems approaches to the biosciences (experimental biology closely-integrated with computational modeling).
 
Title 3D-printed dynamic models of DNA and transcription factors 
Description Using rapid prototyping, we have created a new type of educational tools to explain, through tactile interaction, the mechanical properties of double-stranded DNA and the mechanism of gene regulation. We believe that this is currently the only method to explain complex molecular biological precesses to learners with visual disabilities. 
Type Of Art Artefact (including digital) 
Year Produced 2015 
Impact We have used the models to demonstrate the basics of molecular biology at a study group at VISTA, the Leicester society for the blind. 
 
Description We proposed to visualize the process of switching of human genes (i.e. transcription initiation) at the level of single molecules. Transcription initiation is extremely complex, and requires a concerted action of five protein 'nanomachines', called TFIID, TFIIB, TFIIF, TFIIE, and TFIIH. The enzyme that performs the actual initiation, assisted by the five nanomachines, is called RNA polymerase II (RNAP II). Visualization of this process in real time, at the level of single molecules has never been achieved before.

1. During the first year of funding, we built and calibrated a single-molecule super-resolution microscope required for the experiments, which we called the LESTAscope. Using the LESTAScope, we acquired data on single-molecule dynamics of one of the key gene-switching nanomachines -- TFIIF. We have discovered that the conventional view of how TFIIF works was incorrect. In the old model, it was assumed that TFIIF binds to a gene in a single step, once in 15 minutes, already pre-associated with RNAP II. Instead, at the single-molecule level, we have observed that TFIIF interacts with the target gene dozens of times, on a time scale of once in ~5 seconds.

2a. In the second year of funding, we have correlated the gene interactions of TFIIF with switching of target genes. Contrary to predictions of classical models, we have discovered that TFIIF functions, primarily, during the late stages of gene-switching, possibly, by assisting RNAP II in reading through pauses in genes.

2b. In addition, during the second year, we have characterized the single-molecule behavior of another transcription factor, called TFIIB. Similar to the story with TFIIF, TFIIB was predicted to interact with genes only once per gene-switching cycle. However, our single-molecule imaging experiments have revealed that TFIIB interacted with a gene dozens of times before it manages to lock onto the gene -- in a manner entirely consistent with the behavior of TFIIF.

2c. Intrigued with the dynamic behavior of human 'nanomachines' on genes, during the second year, we asked if the behavior could also be observed for another type of human transcription machinery -- the one involved in switching of genes in human mitochondria. Thus, we have reconstructed the mitochondrial transcription machinery under the LESTAscope, and correlated interactions of a mitochondrial transcription factor TFAM with initiation of transcription by mitochondrial RNA polymerase (mtRNAP). Strikingly, we have discovered that, just like we previously observed for TFIIB and TFIIF, TFAM appeared to interact with mitochondrial genes dynamically, through multiple attempts. Therefore, the dynamic aspects of transcription initiation appears to be universal for different types of human RNA polymerases.

3a. During the third year of funding, we have used gene editing by CRISPR/Cas9 to generate new human fibroblast HCT116 cell lines expressing endogenous general transcription factors IID and IIH, in which a key subunit was fused with the genetically encoded protein tag Halo. We purified the tagged IID and IIH using affinity chromatography, fluorescently labeled them, and confirmed the transcription activity of the isolated factors using in vitro transcriptional assays. This will allow us imaging assembly of the full pre-initiation complex, now with IID and IIH, in vitro and, in future research, in living cells.

3b. In addition, during the third year of funding, we have adapted an entirely new strategy towards fluorescence tagging and single-molecule analysis of dynamics of recombinant human general transcription factors: IIB, IIF, and IIE. In the past, we relied on cysteine-based labeling to generate fluorescently tagged versions of these proteins. Although this has allowed us to get first preliminary data on the dynamic behavior of IIF and IIB on promoters, this method favors labeling of unstructured, disordered transcription factors molecules whose behavior may not faithfully reflect that of native factors. In addition, cysteine-based labeling would not permit imaging of the activity of these factors in living cells. To circumvent this problem, we have generated a new panel of N- and C-terminal Halo-tag fusions of the transcription factor IIB, and subunits of IIF and IIE. All of the purified and labeled factors have retained their activity in vitro.

3c. In addition, during the third year of funding, we have started working on a new DNA origami-based technology that would allow us to probe the dynamic interactions of the transcription 'nanomachines' with genes in cells. This level of sensitivity has never been achieved before, due to the basic laws of physics that dictate that objects smaller than the wavelength of light (about 500 nm) cannot be resolved with light microscopy. To circumvent this, we have created self-assembled nanostructures intended to harness the electromagnetic properties of light to achieve focusing 'far beyond' the 500 nm limit, (i.e. the size of a promoter or a single transcription factor molecule). During the third and the fourth year, we have fabricated the nanostructures, and found a UV-based approach to make the nanostructures robust and resistant to laser irradiation.

4a. During the fourth year, we have put together all the technological advancements we made during years 1-3, and , for the first time and in real-time, visualized initiation of transcription, promoter escape, and elongation by RNAPII. The experiments were carried out using fluorescently labeled real-time RNA sensor, labeled RNAPII, and labeled IIH (created using CRISPR technology in year 2). We are currently working on final control experiments for manuscript submission (possible journals: Cell, Genes and Development).

4b. In addition, during the fourth year, we have finished the story on single-molecule real-time visualization of initiation by human mtRNAP, and are currently preparing the work for publication (possible journals: Cell, Molecular Cell, PNAS). The work has provided the first comprehensive single-molecule view of the full transcription cycle by mtRNAP, including the measurements of rates of transcription initiation, escape, and elongation, as well as the demonstration of promoter scanning by the transcription factor TFAM. The most striking finding was that mtRNAP rides on TFAM during promoter scanning by TFAM, a phenomenon which could only be observed by single-molecule techniques. We believe that the final promoter recognition step is carried out by the transcription factor TFB2M, which enters the preinitiation complex right before promoter escape. To confirm this, during the fourth year, we have developed a new fluorescent label (which we call the TripleHalo tag) which will allow us to carry out three-colour single-molecule imaging experiments using labeled TFAM, labeled mtRNAP, labeled TFB2M, and labeled real-time RNA sensor.

4c. In addition, during the fourth year, we have discovered that laser-induced photodamage during continuous fluorescence imaging (which requires laser power densities equivalent to sunlight density multiplied hundred- to thousand-fold) greatly reduces the transcription activity of human RNA polymerase II. This issue of photodamage (which is not the same as the better-known issue of photobleaching) has been largely overlooked in the field (including our own lab). Although we have addressed the issue of photodamage in the short-term (by fabricating a self-contained inert gas chamber, see below), a long-term solution would need to be found in the field, for instance, nanofocusing of light (see 3c above).

4d. During the last critical three months of funding, we have experienced an unexpected delay in research due to an action taken by our business management which has resulted in the loss of a precision workshop, which, as stated in our original application, was central to this research. Although the workshop was an asset of our University, the action was taken despite the appeals from the academics. Thus, to compensate for the workshop loss, we had to invest our time to set up our own independent workshop at the Revyakin lab, and the PI had to fabricate all the required parts himself (the inert-gas chamber required for photodamage control).

5a. In the fifth year since the award was granted, we have reconstructed the full transcription initiation by human transcription factor IIB and fluorescently tagged human RNA polymerase II, at single-molecule resolution. We have discovered that, contrary to accepted 'textbook' model of human transcription initiation, human transcription factor IIB and Pol2 pre-associated together prior to initiating transcription (in at least 50% of observed events). Upon promoter escape by RNA polymerase II, TFIIB gets released within 5 seconds. We have reported this discovery at the International Biophysical Society meeting in San Diego (February, 2020). The dynamic behavior is consistent with TFIIB acting as a human functional analog of the bacterial transcription initiation factor sigma. Sigma is known to pre-associate with the bacterial RNA polymerase and to form the so-called holoenzyme complex with the polymerase.

5b. In addition, in the fifth year since the award was granted, we have benchmarked the new TripleHalo fluorescent tag (see 4b above) using biochemical gel-shift and real-time single-molecule binding assays. As we expected, the tag provided a considerable (2.5+) boost of photon counts which will enable us to create fluorescent tags in spectral bands previously under-utilized in single-molecule analysis (the 'cyan', 500-520 nm band and the near infrared, 750-800 nm band).

5c. In addition, in the fifth year since the award was granted, we have further developed the strategy to generate robust DNA origamis, using a novel psoralen/hoogsteen helix-based crosslinking strategy.

5d. Finally, in the fifth year since the award was granted, we have focused on tagging of the most mysterious transcription factor of the human mitochondrial transcription machinery - TFB2M -- to permit the visualization of its single-molecule dynamics. We have so far tried generating terminal and internal fusions of the protein with the Halo and SNAP tags, both of which, disappointingly, produced non-functional proteins. Our current strategy is to employ cysteine tagging and unnatural aminoacids to generate fluorescently labeled TFB2M.
Exploitation Route The LESTASCope can be directly used as a super-resolution platform for in vitro and in vivo imaging of dynamic biological assembly. At Leicester University, we are collaborating with the lab of Prof. Ruth-Carter to visualize micro-RNA in cell culture models of Huntington's disease. There is also considerable interest at our University to use the instrument to study co-transcriptional splicing (Dr. O. Makarova and Prof. I. Eperon), actin filament organization (Dr. M. El-Mezgueldi), cell division in yeast (Dr. Tanaka), and histone deacetylase complexes (Prof. J. Schwabe). In addition, we have started a collaboration with the group of Dr. M. Dahan (Marie Curie Institute, Paris, France) to visualize single-molecule interactions between transcription factors derived from plants (TALe) and their target genes.

Our nanostructure-based technology can be used for single-molecule functional and structural analysis of protein complexes that carry out nucleic acid transactions. In addition, our nanostructures can be used as structural markers and display molecules for EM tomography in highly heterogenous and crowded mixtures (e.g. nuclear extracts and even cell slices). In addition, the nanofocusing devices can be used for benchtop low-cost single-molecule DNA sequencing (currently, state-of-the-art single-molecule sequencing machines by Pacific Biosciences cost ~1 mln GBP). Finally, the technology can be used for making solar cells and for fabrication of P/N junctions in transistors.

The glass surface passivation technology that we adapted to immobilize single genes in the field of view of the LESTAScope can be used to prepare bio-compatible fluidic devices for high-throughput screening.

The LESTAScope and nanostructure-based imaging platforms can be both adapted for high-throughput chemical screening, particularly to search for new drugs that affect gene regulation in eukaryotic and bacterial cells.

The TripleHalo tag is very likely to generate considerable interest in the fluorscence-imaging community. I intend to make this available to the reserach community with no licensing fees and no adminitrative costs.

The stable psoralen-based DNA origamis are likely to transform the DNA origami nanotechnology field, currently lacking reproducible, specific methods to produce stable nanostructures. I intend to make this available to the reserach community with no licensing fees and no adminitrative costs.
Sectors Chemicals,Construction,Education,Electronics,Energy,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description As outlined in the original Pathways to Impact for this Award, one of the Impact Objectives (Objective 2.1) was to increase the public awareness of our research on gene regulation. Understanding how gene-regulatory protein molecules interact in space and time to switch genes on is the primary goal of our research. However, bringing our findings to the general public (like any finding in basic molecular biology) is not trivial: it often takes decades of dedicated work to comprehend the complexity of the molecular world of genes and proteins. The most common method is to use highly simplified computer animations and/or statics diagrams. However, these cannot possibly capture the stochastic behaviour of regulatory molecules, which, as we are now discovering, is in the core of gene regulation (for example, the random promoter search by the protein TFIIF). In addition, learners with visual disabilities cannot possible benefit from commonly used visual demonstrations and diagrams. Thus, we have now designed and produced tactile 3D models of genes and gene-regulatory proteins which we already used to demonstrate basic concepts of gene regulation to local students and to visually impaired members of the general public. The main focus of the demonstrations was the stochastic/random behaviour of molecules, and how a seemingly random sequence of molecular events can lead to complex cell behaviour. In October of 2016, AR has participated in the University of Leicester Open Day event and used the hands-on 3D models in his lecture entitled "Seeing molecules switching genes in a cell, in a galaxy far-far away". In the lecture, AR drew parallels between technologies used to detect alien life on distant planets and to detect protein-DNA interactions in living cells. Following this event, in November of 2017, AR has sponsored a day-long visit by an A-levels student in which the student carried out labelling of the human transcription factor IIF using fluorescent dyes. In March, 2018, AR was invited to give a 1-hour long lecture at the headquarters of the pharmaceutical giant Norvartis in Basel, Switzeland, together with a small selected group of experts on human transcription regulation (10 researchers from the USA, UK, and Europe). The goal of the meeting was to present hot research foci and emerging technologies in the field of gene transcription, and to advice the company on challenges in development of drugs targeting human transcription regulators. In October of 2018, AR has given a lecture at a University of Leicester Open Day in which he made high-impact demonstrations on the use of fluorescent chemicals in daily life.
First Year Of Impact 2014
Sector Communities and Social Services/Policy,Education,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic
 
Description Bio-imaging Review: BBSRC group of Critical Friends
Geographic Reach National 
Policy Influence Type Participation in a guidance/advisory committee
 
Description NVIDIA equipment fund
Amount £4,000 (GBP)
Organisation NVIDIA 
Sector Private
Country Global
Start 10/2015 
End 10/2015
 
Title Cloudy PEG-based passivation method for glass surfaces for single-molecule imaging 
Description This effective, robust protocol generates glass coverslips coated with biotin-functionalized polyethylene glycol (PEG), making the glass surface resistant to non-specific absorption of biomolecules, and permitting immobilization of biomolecules for subsequent single-molecule tracking of biochemical reactions. The protocol can be completed in one day, and the coverslips can be stored for at least 1 month. We have confirmed that the PEG surfaces prepared according to the protocol are resistant to non-specific adsorption by a wide range of biomolecules (bacterial, mitochondrial, and human transcription factors, DNA, and RNA) and biological buffers. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact To maximise the adoption of the method by other researchers, we published the protocol in a free on-line publication (Bio-Protocol), and provided most detailed instructions to make the surface passivation reproducible 
 
Title Human Cell lines expressiong fluorescently tagged endogenous general transcription factors, TFIID and TFIIH 
Description One of the challenges in single-molecule analysis of dynamic biological processes, such as assembly of transcription complexes, is putting fluorescent dyes on multi-subunit protein complexes that are not available in recombinant form. During the year of 2017, Oksana Gonchar has used gene editing by CRISPR/Cas9 to generate human fibroblast HCT116 cell lines expressing endogenous general transcription factors IID and IIH in which a key subunit was fused with the genetically encoded protein tag Halo. She has purified the tagged IID and IIH using affinity chromatography, fluorescently labeled them, and confirmed the transcription activity of the isolated factors using in vitro transcriptional assays. This will allow us imaging assembly of the full pre-initiation complex, now with IID and IIH, in vitro and, in future research, in living cells. 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? No  
Impact Not yet 
 
Title LESTASCOPE: a new single-molecule super-resolution microscope 
Description During the first year of funding, we have designed and built LESTAscope, a state-of-the-art four colour single-molecule microscope which is optimized to visualize the assembly and function of macromolecular complexes in real-time, at high throughput (~2,000 assembly sites simultaneously). The instrument is actively stabilized in three dimensions, which permits the use in biological laboratories, at temperatures optimal for enzymes and cells. There is currently no analogues to this instrument in the UK, and, possibly, in the world. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact We are currently collaborating with the Luthi-Carter group at the University of Leicester, to use LESTASCOPE for super-resolution imaging of micro-RNA in neurons which will lead to new insights into the mechanism of Huntington's disease. 
 
Title PEGylated DNA analogs for surface-based assays of DNA-binding proteins 
Description One of main challenges of surface-based enzymatic assays is the effect of non-specific enzyme-surface interactions, due to incomplete/poorly controlled surface modification, and due to the high local concentration of the immobilized enzyme. This problem is particularly relevant for analysis of protein-DNA intractions using surface-immobilized DNA. To circumvent this problem, we have created a method for generation of PEGylated DNA fragments that protect the enzymes interacting with the DNA from detrimental surface effects, while preserving the affinity of the enzymes to the DNA elements of interest. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact The method has not yet been disseminated. Once published, the method can be used for single-molecule analysis of protein-DNA interactions in vitro, for passivation of DNA nanostructures, and for high-throughput surface-based screening of small molecules targeting transcription factors 
 
Title TripleHalo protein tag 
Description We have developed a new recombinant protein tag for generating labeled fusion proteins -- TripleHalo tag. TripleHalo is a fusion of three HaloTags head-to-tail (total additional Mw about 90 kDa). When expressed as a fusion with a protein of interest, TripleHalo permits labeling of the protein with up to three fluorescent dye residues. This may potentially allow encoding of protein molecules in single-molecule assays using dye intensity, thus expanding the palette of imaging colours. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? No  
Impact Once published, this reagent will be distributed to the research community. 
 
Description Design and fabrication of light-focussing DNA nanostructures for single-molecule imaging of transcription initiation 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution Design of promoter-containing DNA nanostructures for single-molecule imaging of transcription initiation
Collaborator Contribution Dr Dmitry Cherny at the University of Leicester provided Expertise in Transmission Electron Microscopy (negative staining, data collection, carbon grid preparation, and data analysis)
Impact This is an ongoing collaboration to create new DNA nanostructures which would serve as platforms for assemble of human RNA Polymerase II pre-initiation complexes. Assembly of proteins on DNA nanostructures is expected to eliminate surface artefacts and to provide a scaffold for assembly of light-harvesting plasmonic nano-antennas. The latter would allow, for the first time, single-molecule imaging of transcription initiation in the crowded environment of the human cell.
Start Year 2016
 
Description Design of plasmonic nanostructures to focus light onto single promoters; collaboration with the group of Alexander Krasnok. 
Organisation ITMO University
Department Department of Nano-Photonics and Metamaterials
Country Russian Federation 
Sector Academic/University 
PI Contribution We came up with the idea to use gold bowtie nano-antennas to focus visible light into volumes large enough to accommodate transcription initiation complexes on promoters, and yet small enough to permit single-molecule imaging of assembly of these complexes at physiologically relevant protein concentrations.
Collaborator Contribution The group of Alexander Krasnok has carried out FDTD simulations of electromagnetic fields to identify the optimal shape of bowtie nano-antennas suitable for focusing light onto single promoter molecules, without affecting the transcription initiation process Furthermore, they have nano-fabricated prototype structures that we will test in single-molecule imaging conditions using our recently built instrument, the LESTAScope.
Impact None yet.
Start Year 2015
 
Description Outreach to disseminate knowledge on gene regulation 
Organisation Genetics Education Networking for Innovation and Excellence (GENIE)
Country United Kingdom 
Sector Academic/University 
PI Contribution We have designed and produced 3D models of gene fragments and transcription factors, which are now used by the GENIE outreach team at the University of Leicester (lead by Dr. Cas Kramer) to teach Leicester University students and Leicestershire middle school students the basics of gene regulation.
Collaborator Contribution Dr. Cas Kramer has provided his expertise in outreach to help us design 3D structural models of protein and DNA that work best in demonstrations. He then tested our models in teaching sessions, and provided feedback.
Impact We are considering to carry out a research study to compare the effectiveness of 3D physical models to the effectiveness of conventional computer visualization in teaching the basics of protein-DNA interactions.
Start Year 2014
 
Description Single-molecule analysis of interactions of TALEs with cognate DNA sequences: Maxime Dahan's group, Marie Curie Institute, France 
Organisation Curie Institute Paris (Institut Curie)
Country France 
Sector Academic/University 
PI Contribution We have provided the expertise in single-molecule real-time imaging of assembly of macromolecular complexes on immobilised DNA, and data analysis.
Collaborator Contribution The Dahan group provided the biological question for this collaboration, specifically, how do TALE proteins recognise their target DNA sequences in living cells. The Dahan group has further provided the expertise in purifying and measuring the activity of TALE proteins and interpretation of data.
Impact This is a multidisciplinary collaboration at the interface of Optical Physics, Cell Biology, Surface Chemistry, and Biochemistry.
Start Year 2017
 
Description Super-resolution mapping of micro-RNA in cell culture models of Huntington's disease 
Organisation Baylor College of Medicine
Department Department of Neuroscience
Country United States 
Sector Academic/University 
PI Contribution We have provided out expertise in single-molecule imaging, and our expertise in designing and making of hybridization probes to visualize micro-RNA at the single-molecule level.
Collaborator Contribution The group of Prof. Luthi-Carter has identified a new species of micro-RNA that might be associated with the progression of Huntington's disease. They have designed sequences of probes to detect the micro-RNA in cell culture models of Huntington's disease.
Impact Not outputs yet. The preliminary data on micro-RNA localization will be used to apply for dedicated funding.
Start Year 2015
 
Description BBSRC Review of Bioimaging by Critical Group of Friends 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact A.R. is currently serving on a panel of ~20 participants, selected by the BBSRC from UK experts in bio-imaging, to analyse the BBSRC bio-imaging portfolio (i.e. to identify which areas of bio-imaging are over-, or under-represented in the portfolio, and which areas are likely to be 'hot' in the next 10 years). As future directions, the panel has so far identified multi-disciplinarity, increased time/space resolution, improved imaging processing algorithms, in-vivo/in-organism imaging, and synthesis of new imaging probes.
Year(s) Of Engagement Activity 2015,2016
 
Description Dynamic DNA outreach event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 100+ pupils from Leicestershire schools have visited the University of Leicester, to participate in the Dynamic DNA outreach event. During this event, the pupils participated in hands-on activities to learn the basics of genetics, molecular biology and biochemistry.

My research group has produced 3D models of DNA base-pairs shaped in the form of USB thumbdrives. These were distributed among students as awards for the best drawing of Drosophila mutants.
Year(s) Of Engagement Activity 2015
URL http://www2.le.ac.uk/departments/genetics/genie
 
Description Structural biology for the visually impaired, together with the VISTA society for the blind 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Understanding the complex shapes of biological molecules requires sophisticated computer visualization software. For the visually impaired, however, there is currently no tools available to visualize molecular shapes. Our research group has used a 3D printer to design and produce 3D models of DNA and proteins which could be used as molecular biologist' 'Braile' to explain basic concepts of molecular biology to the visual impaired. To test the models, we have visited a group of members of the VISTA, the Leicester society for the blind, and brought with us 3D models of DNA and transcription factors. The meeting lasted for about 1 hr, and we are currently organizing a bigger event.
Year(s) Of Engagement Activity 2016
 
Description University of Leicester Open Day, Autumn 2016 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 106 Year 12 students and 10 teachers attended the department and participated in hands-on activities and tours and attended a series of lectures on diverse topics having to do with gene expression. A.R. has delivered a 30-min long lecture entitled "Seeing molecules switching genes in a cell, in a galaxy far-far away". In the lecture, AR drew parallels between technologies used to detect alien life on distant planets and to detect protein-DNA interactions in living cells. Following this event, in November of 2017, AR has sponsored a day-long visit by an A-levels student in which the student carried out labeling of the human transcription factor IIF using fluorescent dyes.
Year(s) Of Engagement Activity 2016