Imperial College London

Professor David S. Rueda

Faculty of MedicineDepartment of Infectious Disease

Chair in Molecular and Cellular Biophysics
 
 
 
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Contact

 

david.rueda Website

 
 
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Location

 

6.12DLMS BuildingHammersmith Campus

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Summary

 

Publications

Publication Type
Year
to

133 results found

Masood M, Ding Q, Cawte AD, Rueda DS, Grimm SW, Yagüe E, El-Bahrawy Met al., 2023, Genetic screening for anticancer genes highlights FBLN5 as a synthetic lethal partner of MYC, Cell Communication and Signaling, Vol: 21, ISSN: 1478-811X

Background: When ectopically overexpressed, anticancer genes, such as TRAIL, PAR4 and ORCTL3, specifically destroy tumour cells without harming untransformed cells. Anticancer genes can not only serve as powerful tumour specific therapy tools but studying their mode of action can reveal mechanisms underlying the neoplastic transformation, sustenance and spread. Methods: Anticancer gene discovery is normally accidental. Here we describe a systematic, gain of function, forward genetic screen in mammalian cells starting with over 30,000 transcripts in order to isolate novel anticancer genes of human origin. FBLN5 was chosen, as a proof of principle, for mechanistic gene expression profiling, comparison pathways analyses and functional studies. Results: Sixteen novel anticancer genes were isolated; these included non-coding RNAs, protein-coding genes and novel transcripts, such as ZNF436-AS1, SMLR1, TMEFF2, LINC01529, HYAL2, NEIL2, FBLN5, YPEL4 and PHKA2-processed transcript. FBLN5 selectively caused inhibition of MYC in COS-7 (transformed) cells but not in CV-1 (normal) cells. MYC was identified as synthetic lethality partner of FBLN5 where MYC transformed CV-1 cells experienced cell death upon FBLN5 transfection, whereas FBLN5 lost cell death induction in MCF-7 cells upon MYC knockdown. Conclusions: Sixteen novel anticancer genes are present in human genome including FBLN5. MYC is a synthetic lethality partner of FBLN5.

Journal article

Ploetz E, Ambrose B, Barth A, Börner R, Erichson F, Kapanidis AN, Kim HD, Levitus M, Lohman TM, Mazumder A, Rueda DS, Steffen FD, Cordes T, Magennis SW, Lerner Eet al., 2023, A new twist on PIFE: photoisomerisation-related fluorescence enhancement., Methods Appl Fluoresc, Vol: 12

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate ofcis/transphotoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule. In this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turning PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Journal article

Newton MD, Losito M, Smith QM, Parnandi N, Taylor BJ, Akcakaya P, Maresca M, van Eijk P, Reed SH, Boulton SJ, King GA, Cuomo ME, Rueda DSet al., 2023, Negative DNA supercoiling induces genome-wide Cas9 off-target activity., Mol Cell, Vol: 83, Pages: 3533-3545.e5

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.

Journal article

Han Z, Moore GA, Mitter R, Lopez Martinez D, Wan L, Dirac Svejstrup AB, Rueda DS, Svejstrup JQet al., 2023, DNA-directed termination of RNA polymerase II transcription., Mol Cell, Vol: 83, Pages: 3253-3267.e7

RNA polymerase II (RNAPII) transcription involves initiation from a promoter, transcriptional elongation through the gene, and termination in the terminator region. In bacteria, terminators often contain specific DNA elements provoking polymerase dissociation, but RNAPII transcription termination is thought to be driven entirely by protein co-factors. We used biochemical reconstitution, single-molecule studies, and genome-wide analysis in yeast to study RNAPII termination. Transcription into natural terminators by pure RNAPII results in spontaneous termination at specific sequences containing T-tracts. Single-molecule analysis indicates that termination involves pausing without backtracking. The "torpedo" Rat1-Rai1 exonuclease (XRN2 in humans) greatly stimulates spontaneous termination but is ineffectual on other paused RNAPIIs. By contrast, elongation factor Spt4-Spt5 (DSIF) suppresses termination. Genome-wide analysis further indicates that termination occurs by transcript cleavage at the poly(A) site exposing a new 5' RNA-end that allows Rat1-Rai1 loading, which then catches up with destabilized RNAPII at specific termination sites to end transcription.

Journal article

Belan O, Greenhough L, Kuhlen L, Anand R, Kaczmarczyk A, Gruszka DT, Yardimci H, Zhang X, Rueda DS, West SC, Boulton SJet al., 2023, Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein, Molecular Cell, Vol: 83, Pages: 2925-2940.e8, ISSN: 1097-2765

Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.

Journal article

Aristodemou AENR, Rueda D, Taylor GR, Bangham CRM, Green PLRet al., 2023, The transcriptome of HTLV-1-infected primary cells following reactivation reveals changes to host gene expression central to the proviral life cycle, PLoS Pathogens, Vol: 19, ISSN: 1553-7366

Infections by Human T cell Leukaemia Virus type 1 (HTLV-1) persist for the lifetime of the host by integrating into the genome of CD4+ T cells. Proviral gene expression is essential for proviral survival and the maintenance of the proviral load, through the pro-proliferative changes it induces in infected cells. Despite their role in HTLV-1 infection and a persistent cytotoxic T lymphocyte response raised against the virus, proviral transcripts from the sense-strand are rarely detected in fresh cells extracted from the peripheral blood, and have recently been found to be expressed intermittently by a small subset of cells at a given time. Ex vivo culture of infected cells prompts synchronised proviral expression in infected cells from peripheral blood, allowing the study of factors involved in reactivation in primary cells. Here, we used bulk RNA-seq to examine the host transcriptome over six days in vitro, following proviral reactivation in primary peripheral CD4+ T cells isolated from subjects with non-malignant HTLV-1 infection. Infected cells displayed a conserved response to reactivation, characterised by discrete stages of gene expression, cell division and subsequently horizontal transmission of the virus. We observed widespread changes in Polycomb gene expression following reactivation, including an increase in PRC2 transcript levels and diverse changes in the expression of PRC1 components. We hypothesize that these transcriptional changes constitute a negative feedback loop that maintains proviral latency by re-deposition of H2AK119ub1 following the end of proviral expression. Using RNAi, we found that certain deubiquitinases, BAP1, USP14 and OTUD5 each promote proviral transcription. These data demonstrate the detailed trajectory of HTLV-1 proviral reactivation in primary HTLV-1-carrier lymphocytes and the impact on the host cell.

Journal article

Belan O, Sebald M, Adamowicz M, Anand R, Vancevska A, Neves J, Grinkevich V, Hewitt G, Segura-Bayona S, Bellelli R, Robinson HMR, Higgins GS, Smith GCM, West SC, Rueda DS, Boulton SJet al., 2022, POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells., Mol Cell, Vol: 82, Pages: 4664-4680.e9

POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.

Journal article

Moore G, Han Z, Xu J, Oh J, Wang D, Svejstrup J, Rueda Det al., 2022, Dynamic Backtracking Regulates Lesion Bypass by RNAPII

<jats:title>Abstract</jats:title> <jats:p>The eukaryotic genome is prone to a high amount of DNA damage from intrinsic and extrinsic sources, causing transcriptional stress, including pausing, backtracking and stalling. If not rectified in time, these damages can further lead to transcriptional arrest and genome instability. Here, we develop a single-molecule FRET based elongation complex which allows us to insert various types of DNA damage into the transcribed region and study the effect they have on the dynamics of RNAPII transcription. We show that different DNA lesions cause a heterogenous effect on RNAPII. In some instances, such as oxidative lesions, RNAPII exhibits a high level of dynamic behaviour often backtracking up to 10 nt. While other damages, such as cyclo-butane pyrimidine dimers and abasic sites, can cause more significant static stalling. Furthermore, the repair factor Rad26 binds to RNAPII and alters these dynamics by pushing RNAPII further over the damage site and preventing long-range backtracking events.</jats:p>

Journal article

Kaczmarczyk AP, Déclais A-C, Newton MD, Boulton SJ, Lilley DMJ, Rueda DSet al., 2022, Search and processing of Holliday junctions within long DNA by junction-resolving enzymes, Nature Communications, Vol: 13, Pages: 1-13

Resolution of Holliday junctions is a critical intermediate step of homologous recombination in which junctions are processed by junction-resolving endonucleases. Although binding and cleavage are well understood, the question remains how the enzymes locate their substrate within long duplex DNA. Here we track fluorescent dimers of endonuclease I on DNA, presenting the complete single-molecule reaction trajectory for a junction-resolving enzyme finding and cleaving a Holliday junction. We show that the enzyme binds remotely to dsDNA and then undergoes 1D diffusion. Upon encountering a four-way junction, a catalytically-impaired mutant remains bound at that point. An active enzyme, however, cleaves the junction after a few seconds. Quantitative analysis provides a comprehensive description of the facilitated diffusion mechanism. We show that the eukaryotic junction-resolving enzyme GEN1 also undergoes facilitated diffusion on dsDNA until it becomes located at a junction, so that the general resolution trajectory is probably applicable to many junction resolving enzymes.

Journal article

Kaczmarczyk A, Déclais A-C, Newton M, Boulton S, Lilley D, Rueda Det al., 2022, Search and Processing of Holliday Junctions within Long DNA by Junction-Resolving Enzymes

<jats:title>Abstract</jats:title> <jats:p>Resolution of four-way Holliday junctions is a critical intermediate step of homologous recombination. DNA junctions must be processed by abundant junction-resolving endonucleases to cleave the covalent link between two chromosomes. Although the catalytic activity and interaction mode of endonucleases with Holliday junction have been characterized in great detail by biochemical and structural studies, it remains unclear how the enzymes find their substrate located within kilobase pairs of duplex DNA. Here, we present a complete single-molecule reaction trajectory for a junction-resolving enzyme finding and cleaving a Holliday junction. We employed correlative optical tweezers with confocal fluorescence microscopy to track a single dimer of endonuclease I on the DNA template in real-time. We observed that the enzyme binds remotely to the dsDNA and then undergoes 1D diffusion. Upon encountering the four-way junction, a catalytically impaired endonuclease I mutant remains bound at that point for long periods. An active enzyme, however, cleaves the junction after a few seconds. When an N-terminal truncated endonuclease mutant is used, the enzyme fails to diffuse along dsDNA and dissociates after short periods of time, indicating that DNA encirclement by the disordered N-terminus is a key requirement for 1D diffusion and efficient target localisation. Quantitative analysis of each of these stages revealed a comprehensive description of the facilitated diffusion mechanism. We show that the eukaryotic junction-resolving enzyme GEN1 also undergoes facilitated diffusion on dsDNA until it becomes located at a junction, so that the general resolution trajectory is likely to be applicable to most junction resolving enzymes.</jats:p>

Working paper

Poudevigne-Durance P, Hug N, Mukhortava A, Caceres J, Rueda DSet al., 2022, Single-molecule RNA remodelling by the DExH-Box 34 helicase, Publisher: CELL PRESS, Pages: 481A-481A, ISSN: 0006-3495

Conference paper

Newton MD, Taylor BJ, Cuomo ME, Rueda DSet al., 2022, CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy., Methods Mol Biol, Vol: 2478, Pages: 349-378

The discovery of CRISPR/Cas9 as an easily programmable endonuclease heralds a new era of genetic manipulation. With this comes the prospect of novel gene therapy approaches, and the potential to cure previously untreatable genetic diseases. However, reports of spurious off-target editing by CRISPR/Cas9 pose a significant hurdle to realizing this potential. A deeper understanding of the factors that affect Cas9 specificity is vital for development of safe and efficient therapeutics. Here, we describe methods for the use of optical tweezers combined with confocal fluorescence microscopy and microfluidics for the analysis of on- and off-target activity of Cas9 activity.

Journal article

Anand R, Buechelmaier E, Belan O, Newton M, Vancevska A, Kaczmarczyk A, Takaki T, Rueda DS, Powell SN, Boulton SJet al., 2021, HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51, Nature, Vol: 601, Pages: 268-273, ISSN: 0028-0836

DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2,3,4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.

Journal article

Rueda D, Cawte AD, Ino H, Unrau Pet al., 2021, Single-molecule RNA Imaging Using Mango II Arrays, Methods in Molecular Biology, Publisher: Humana Press

In recent years, fluorogenic RNA aptamers, such as Spinach, Broccoli, Corn, Mango, Coral, and Pepper have gathered traction as an efficient alternative labeling strategy for background-free imaging of cellular RNAs. However, their application has been somewhat limited by relatively inefficient folding and fluorescent stability. With the recent advent of novel RNA-Mango variants which are improved in both fluorescence intensity and folding stability in tandem arrays, it is now possible to image RNAs with single-molecule sensitivity. Here we discuss the protocol for imaging Mango II tagged RNAs in both fixed and live cells.

Book chapter

Newton MD, Fairbanks SD, Thomas JA, Rueda DSet al., 2021, A Minimal Load‐and‐Lock Ru II Luminescent DNA Probe, Angewandte Chemie, Vol: 133, Pages: 21120-21127, ISSN: 0044-8249

Journal article

Losito M, Smith QM, Newton MD, Cuomo ME, Rueda DSet al., 2021, Cas12a target search and cleavage on force-stretched DNA, PHYSICAL CHEMISTRY CHEMICAL PHYSICS, Vol: 23, ISSN: 1463-9076

Journal article

Newton MD, Fairbanks SD, Thomas JA, Rueda DSet al., 2021, A minimal load-and-lock Ru-II luminescent DNA probe, Angewandte Chemie International Edition, Vol: 60, Pages: 20952-20959, ISSN: 1433-7851

Threading intercalators bind DNA with high affinities. Here, we describe single-molecule studies on a cell-permeant luminescent dinuclear ruthenium(II) complex that has been previously shown to thread only into short, unstable duplex structures. Using optical tweezers and confocal microscopy, we show that this complex threads and locks into force-extended duplex DNA in a two-step mechanism. Detailed kinetic studies reveal that an individual stereoisomer of the complex exhibits the highest binding affinity reported for such a mono-intercalator. This stereoisomer better preserves the biophysical properties of DNA than the widely used SYTOX Orange. Interestingly, threading into torsionally constrained DNA decreases dramatically, but is rescued on negatively supercoiled DNA. Given the “light-switch” properties of this complex on binding DNA, it can be readily used as a long-lived luminescent label for duplex or negatively supercoiled DNA through a unique “load-and-lock” protocol.

Journal article

Boulton S, Anand R, Buechelmaier E, Belan O, Newton M, Vancevska A, Kaczmarczyk A, Rueda D, Powell Set al., 2021, HELQ is a dual function DSB repair enzyme modulated by RPA and RAD51

<jats:title>Abstract</jats:title> <jats:p>DNA double strand breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3’ to 5’ polarity, whose disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2-4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C and, persistence of RAD51 foci upon DNA damage3,5. Notably, HELQ binds to RPA and the RAD51 paralog BCDX2 complex but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here, we report that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry and single-molecule imaging (SMI), we establish that RAD51 forms a co-complex with and strongly stimulates HELQ as it translocates during DNA unwinding. Conversely, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary strands. Finally, we show that HELQ deficiency in cells compromises single-strand annealing (SSA) and microhomology-mediated end joining (MMEJ) pathways and increases long-tract gene conversion tracts (LTGC) during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair by virtue of co-factor dependent modulation of intrinsic translocase and DNA strand annealing activities.</jats:p>

Working paper

Belan O, Moore G, Kaczmarczyk A, Newton MD, Anand R, Boulton SJ, Rueda DSet al., 2021, Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis., STAR Protocols, Vol: 2, Pages: 1-18, ISSN: 2666-1667

Here, we describe a rapid and versatile protocol to generate gapped DNA substrates for single-molecule (SM) analysis using optical tweezers via site-specific Cas9 nicking and force-induced melting. We provide examples of single-stranded (ss) DNA gaps of different length and position. We outline protocols to visualize these substrates by replication protein A-enhanced Green Fluorescent Protein (RPA-eGFP) and SYTOX Orange staining using commercially available optical tweezers (C-TRAP). Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021).

Journal article

Belan O, Barroso C, Kaczmarczyk A, Anand R, Federico S, OReilly N, Newton MD, Maeots E, Enchev RI, Martinez-Perez E, Rueda DS, Boulton SJet al., 2021, Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins, Molecular Cell, Vol: 81, Pages: 1058-1073.e7, ISSN: 1097-2765

Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a “chaperone” to promote 3′ to 5′ filament growth via highly dynamic engagement with 5′ filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.

Journal article

Belan O, Barroso C, Kaczmarczyk A, Anand R, Federico S, O'Reilly N, Newton MD, Enchev R, Martinez-Perez E, Rueda DS, Boulton Set al., 2021, Single-Molecule Investigation of Rad-51 Presynaptic Filament Assembly and the Role of Mediator Proteins, 65th Annual Meeting of the Biophysical-Society (BPS), Publisher: CELL PRESS, Pages: 33A-33A, ISSN: 0006-3495

Conference paper

Iino H, Abdolahzadeh A, Dolgosheina E, Poudevigne-Durance P, Moore G, Girvan P, Kaczmarczyk A, Zhang T, Newton MD, Unrau PJ, Rueda DSet al., 2021, Rapid Clinical Diagnostic Viral Detection with Saliva by a Novel Single Step Nested Mango-NASBA Assay, 65th Annual Meeting of the Biophysical-Society (BPS), Publisher: CELL PRESS, Pages: 196A-196A, ISSN: 0006-3495

Conference paper

Cawte AD, Unrau PJ, Rueda DS, 2020, Live cell imaging of single RNA molecules with fluorogenic Mango II arrays, Nature Communications, Vol: 11, ISSN: 2041-1723

RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to bleached fluorophore replacement. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.

Journal article

Gutierrez-Escribano P, Newton MD, Llauro A, Huber J, Tanasie L, Davy J, Aly I, Aramayo R, Montoya A, Kramer H, Stigler J, Rueda DS, Aragon Let al., 2019, A conserved ATP- and Scc2/4-dependent activity for cohesin in tethering DNA molecules, Science Advances, Vol: 5, Pages: 1-15, ISSN: 2375-2548

Sister chromatid cohesion requires cohesin to act as a protein linker to hold chromatids together. How cohesin tethers chromatids remains poorly understood. We have used optical tweezers to visualize cohesin as it holds DNA molecules. We show that cohesin complexes tether DNAs in the presence of Scc2/Scc4 and ATP demonstrating a conserved activity from yeast to humans. Cohesin forms two classes of tethers: a “permanent bridge” resisting forces over 80 pN and a force-sensitive “reversible bridge.” The establishment of bridges requires physical proximity of dsDNA segments and occurs in a single step. “Permanent” cohesin bridges slide when they occur in trans, but cannot be removed when in cis. Therefore, DNAs occupy separate physical compartments in cohesin molecules. We finally demonstrate that cohesin tetramers can compact linear DNA molecules stretched by very low force (below 1 pN), consistent with the possibility that, like condensin, cohesin is also capable of loop extrusion.

Journal article

Miura M, Dey S, Ramanayake S, Singh A, Rueda DS, Bangham CRMet al., 2019, Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modelling, PLoS Pathogens, Vol: 15, ISSN: 1553-7366

The human T cell leukemia virus HTLV-1 establishes a persistent infection in vivo in which the viral sense-strand transcription is usually silent at a given time in each cell. However, cellular stress responses trigger the reactivation of HTLV-1, enabling the virus to transmit to a new host cell. Using single-molecule RNA FISH, we measured the kinetics of the HTLV-1 transcriptional reactivation in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1+ individuals. The abundance of the HTLV-1 sense and antisense transcripts was quantified hourly during incubation of the HTLV-1-infected PBMCs ex vivo. We found that, in each cell, the sense-strand transcription occurs in two distinct phases: the initial low-rate transcription is followed by a phase of rapid transcription. The onset of transcription peaked between 1 and 3 hours after the start of in vitro incubation. The variance in the transcription intensity was similar in polyclonal HTLV-1+ PBMCs (with tens of thousands of distinct provirus insertion sites), and in samples with a single dominant HTLV-1+ clone. A stochastic simulation model was developed to estimate the parameters of HTLV-1 proviral transcription kinetics. In PBMCs from a leukemic subject with one dominant T-cell clone, the model indicated that the average duration of HTLV-1 sense-strand activation by Tax (i.e. the rapid transcription) was less than one hour. HTLV-1 antisense transcription was stable during reactivation of the sense-strand. The antisense transcript HBZ was produced at an average rate of ~0.1 molecules per hour per HTLV-1+ cell; however, between 20% and 70% of HTLV-1-infected cells were HBZ-negative at a given time, the percentage depending on the individual subject. HTLV-1-infected cells are exposed to a range of stresses when they are drawn from the host, which initiate the viral reactivation. We conclude that whereas antisense-strand transcription is stable throughout the stress response, the HTLV-1 sense-strand reactivati

Journal article

Cawte AD, Unrau PJ, Rueda DS, 2019, Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays

<jats:title>Abstract</jats:title><jats:p>RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to fluorophore exchange. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.</jats:p>

Working paper

Wilson MD, Renault L, Maskell DP, Ghoneim M, Pye VE, Nans A, Rueda DS, Cherepanov P, Costa Aet al., 2019, Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer, Nature Communications, Vol: 10, ISSN: 2041-1723

Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the histone octamer at the site of integration. To clarify the mechanism of DNA looping by integrase, we determined a 3.9 Å resolution structure of the prototype foamy virus intasome engaged with a nucleosome core particle. The structural data along with complementary single-molecule Förster resonance energy transfer measurements reveal twisting and sliding of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA by approximately two base pairs along the histone octamer accommodates the necessary DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for nucleosomal DNA repositioning, bearing unexpected similarities to chromatin remodelers.

Journal article

Bruno L, Ramlall V, Studer RA, Sauer S, Bradley D, Dharmalingam G, Carroll T, Ghoneim M, Chopin M, Nutt SL, Elderkin S, Rueda DS, Fisher AG, Siggers T, Beltrao P, Merkenschlager Met al., 2019, Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system, Nature Immunology, Vol: 20, Pages: 1372-1380, ISSN: 1529-2908

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of Runx transcription factor paralogs with apparent functional redundancy. Here we asked what cell type-specific biologies might be supported by the selective expression of Runx paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional non-equivalence between Runx paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA-binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain, and evolutionary reconstruction suggested convergence of RUNT domain residues towards sub-maximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.

Journal article

Newton MD, Taylor BJ, Driessen RPC, Roos L, Cvetesic N, Allyjaun S, Lenhard B, Cuomo ME, Rueda DSet al., 2019, DNA stretching induces Cas9 off-target activity, Nature Structural and Molecular Biology, Vol: 26, Pages: 185-192, ISSN: 1545-9985

CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.

Journal article

Newton M, Taylor B, Driessen R, Roos L, Cvetesic N, Allyjaun S, Lenhard B, Cuomo ME, Rueda Det al., 2018, DNA stretching induces Cas9 off-target binding and cleavage, Nature Structural and Molecular Biology, ISSN: 1545-9985

CRISPR/Cas9 is a powerful genome editing tool, but spurious off-target edits present a barrier towards therapeutic applications. To understand how CRISPR/Cas9 discrimi-nates between on- and off-targets, we have developed a single-molecule assay com-bining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule FRET and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with 10 mis-matches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (e.g., transcription, replication, etc) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.

Journal article

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