A short peptide that preferentially binds c-MYC G-quadruplex DNA

G-quadruplexes (G4s) are non-canonical DNA secondary structures. The identification of selective tools to probe individual G4s over the ~700,000 found in the human genome is key to unravel the biological significance of specific G4s. We took inspiration from a crystal structure of the bovine DHX36 helicase bound to the G4 formed in the promoter region of the oncogene c-MYC to identify a short peptide that preferentially binds MYC G4 with nM affinity over a small panel of parallel and antiparallel G4s tested

Replication-induced DNA secondary structures drive fork uncoupling and breakage

Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress

Research data in support of "A synthetic signalling network imitating the action of immune cells in response to bacterial metabolism"

This repository contains raw data in support of the publication 10.1002/adma.202301562. Please refer to the published paper, available open access, for full information and context. Figure 2 data - Panel b: Confocal image of core-shell particle - Panel d: Absorbance data to probe pH responsiveness of the DNA constructs - Panel c: CD data to probe pH responsiveness of the DNA constructs - Panel g: Representative epifluorescence and bright field images and bright field videos to demonstrate pH-induced particle aggregation Figure 3 data - Panel b: pH and OD data vs E. coli growth time in various glucose concentrations - Panels d, e; Representative epifluorescence images and bright field videos to demonstrate E. coli induced particle aggregation and bacteria trapping - Panel f: OD data vs E. coli growth time Figure 4 data - Panel b: OD data vs E. coli growth time showing effect of netosis-like synthetic pathway - Panel e: Representative bright field and epifluorescence images showing effect of netosis-like synthetic pathway Supplementary Figure 1: Agarose gel demonstrating DNA construct assembly Supplementary Figure 2: DLS data showing pH responsiveness of DNA nanostructures Supplementary Figure 3: UV melting curve data for DNA constructs Supplementary Figure 4: DLS data underpinning pH response curve Supplementary Figure 5: Representative data provided in Figure 2, Panel g folder Supplementary Figure 6: Representative microscopy videos used to generate hydrodynamic radius data with DDM Supplementary Figure 7: OD data vs E. coli growth time at various glucose concentrations Supplementary Figure 8: FITC dextran fluorescence intensity data (used to determine pH) vs E. coli growth time at various glucose concentrations Supplementary Figure 9: Calibration data (FITC dextran fluorescence vs pH) used in Figure 3f and Supplementary Figures 8 and 12 Supplementary Figure 10: FITC dextran fluorescence intensity data (used to determine pH in Figure 3f) vs E. coli growth time at various glucose concentrations Supplementary Figure 11: Representative data provided in Figure 3, Panel d, e folder Supplementary Figure 12: FITC dextran fluorescence intensity data (used to determine pH) vs E. coli growth time Supplementary Figure 13: Calibration data (Fluorescein dT fluorescence vs pH) for fluorescent pH probe linked to DNA particles Supplementary Figure 14: Fluorescent data for pH-induced de-quenching on DNA particles Supplementary Figure 15: OD vs E. coli growth time at various antibiotic concentrations Supplementary Figure 16: Representative data provided in Figure 4, Panel c, folde

A Synthetic Signaling Network Imitating the Action of Immune Cells in Response to Bacterial Metabolism.

State-of-the-art bottom-up synthetic biology allows to replicate many basic biological functions in artificial-cell-like devices. To mimic more complex behaviors, however, artificial cells would need to perform many of these functions in a synergistic and coordinated fashion, which remains elusive. Here, a sophisticated biological response is considered, namely the capture and deactivation of pathogens by neutrophil immune cells, through the process of netosis. A consortium consisting of two synthetic agents is designed-responsive DNA-based particles and antibiotic-loaded lipid vesicles-whose coordinated action mimics the sought immune-like response when triggered by bacterial metabolism. The artificial netosis-like response emerges from a series of interlinked sensing and communication pathways between the live and synthetic agents, and translates into both physical and chemical antimicrobial actions, namely bacteria immobilization and exposure to antibiotics. The results demonstrate how advanced life-like responses can be prescribed with a relatively small number of synthetic molecular components, and outlines a new strategy for artificial-cell-based antimicrobial solutions

A Synthetic Signalling Network Imitating the Action of Immune Cells in Response to Bacterial Metabolism.

State-of-the-art bottom-up synthetic biology allows us to replicate many basic biological functions in artificial cell-like devices. To mimic more complex behaviours, however, artificial cells would need to perform many of these functions in a synergistic and coordinated fashion, which remains elusive. Here we considered a sophisticated biological response, namely the capture and deactivation of pathogens by neutrophil immune cells, through the process of netosis. We designed a consortium consisting of two synthetic agents - responsive DNA-based particles and antibiotic-loaded lipid vesicles - whose coordinated action mimics the sought immune-like response when triggered by bacterial metabolism. The artificial netosis-like response emerges from a series of interlinked sensing and communication pathways between the live and synthetic agents, and translates into both physical and chemical antimicrobial actions, namely bacteria immobilisation and exposure to antibiotics. Our results demonstrate how advanced life-like responses can be prescribed with a relatively small number of synthetic molecular components, and outlines a new strategy for artificial-cell-based antimicrobial solutions. This article is protected by copyright. All rights reserved

Replication-induced DNA secondary structures drive fork uncoupling and breakage.

Funder: Lister Institute of Preventive Medicine (Lister Institute); doi: http://dx.doi.org/10.13039/501100001255Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress

The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes

Funder: Lister Institute of Preventive Medicine; doi: https://doi.org/10.13039/501100001255AbstractAmyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC)n in C9orf72. Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD.</jats:p

Research data supporting "The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes"

Data repository for work contributing to the paper "The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes". Confocal micrographs are presented as raw .TIFF files with corresponding metadata .txt file, which can be opened using text editing software such as Notepad. Confocal micrographs were collected using a Leica SP8 Inverted Confocal microscope (HC PL APO CS2 10x/0.40 DRY | HC PL APO CS2 20x/0.75 DRY objectives). Circular Dichroism spectra were collected using a Quartz High Precision cell (1 mm light path) and analysed in the Jasco J-715 Spectropolarimeter. The RAW data is saved in .xlsx file which can be accessed in Microsoft Excel or any other txt reader. Gel imaging was performed with Typhoon FLA 9500. The images are saved in a .ppt file which can be opened with Microsoft Powerpoint. Flow Cythometry data was aquired using a BD LSRFortessa analyser operated by the FACSDiva software (BD), and the results were analysed using the FlowJo software. The paper is open access and further details for the data acquisition can be found in the Methods section.RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) - BB/R011605/1 [Di Antonio] Lister Institute of Preventive Medicine [Di Antonio] Leverhulme Trust - EP/S023518/1 [Raguseo] Francis Crick Institute (Francis Crick Institute Limited) - P2022-0007 [Wang] RCUK | MRC | Medical Research Foundation - MR/S006591/1 [Petrić Howe] Academy of Medical Sciences - SGL027\1022 [Balendra] EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council) - 851667 [Tanase] RCUK | Engineering and Physical Sciences Research Council (EPSRC) - G98210 [Maher] EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council) - 851667 [Malouf] RCUK | Engineering and Physical Sciences Research Council (EPSRC) - MR/S033947/1 [Aprile] Alzheimer's Research UK (ARUK) - PG2019B-020 [Aprile] RCUK | Engineering and Physical Sciences Research Council (EPSRC) - MR/S031537/1 [Elani] RCUK | Medical Research Council (MRC) - MR/S006591/1 [Patani] Lister Institute of Preventive Medicine [Patani] EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council) - 851667 [Di Michele

Early Antibody Dynamics in a Prospective Cohort of Children At Risk of Celiac Disease

Introduction: The purpose of this study was to identify possible serum biomarkers predicting celiac disease (CD) onset in children at risk. Methods: A subgroup from an ongoing, international prospective study of children at risk of CD was classified according to an early trajectory of deamidated gliadin peptides (DGPs) immunoglobulin (Ig) G and clinical outcomes (CD, potential CD, and CD autoimmunity). Results: Thirty-eight of 325 children developed anti-tissue transglutaminase IgA antibody (anti-tTG IgA) seroconversion. Twenty-eight of 38 children (73.6%) showed an increase in anti-DGPs IgG before their first anti-tTG IgA seroconversion. Discussion: Anti-DGPs IgG can represent an early preclinical biomarker predicting CD onset in children at risk