Biology subjects

Raguseo, F.

Publications and source records attributed to Raguseo, F..

3 recordsLinked to original sources

bioRxiv2023

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.

synthetic biology↗

bioRxiv2023

The C9Orf72 hexanucleotide repeat expansion transcript forms insoluble aggregates facilitated by multimolecular G-quadruplex structures

Amyotrophic 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 the expansion of the intronic hexanucleotide repeat (GGGGCC)nin C9orf72. Here, we investigated the formation of nucleic-acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of the diseases. We observed significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associated to the formation of multimolecular G-quadruplexes (mG4s), using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions led to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validated the biological relevance of our findings by demonstrating the ability of a G4-selective fluorescent probe to penetrate C9orf72 mutant human motor neurons derived from ALS patients, which revealed clear fluorescent 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 and FTD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/526399v3_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@18c11e6org.highwire.dtl.DTLVardef@eb308eorg.highwire.dtl.DTLVardef@c0c870org.highwire.dtl.DTLVardef@a7555a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

The Response of the Eukaryotic Replisome to G-quadruplex- and i-motif-forming Sequences

Sequences that can form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play a range of physiological roles. However, they can also pose a challenge to the replication machinery and in turn threaten genome stability. Multiple lines of evidence suggest G4s interfere with replication, but the underlying mechanism remains unclear. Moreover, there is a lack of evidence of how iMs affect the replisome. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM is sufficient to arrest DNA replication. Direct single molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. A combination of genetic and biophysical characterisation establishes that structure forming capacity is a key determinant of replisome arrest. Mechanistically, replication fork 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 Sgs1 or Chl1. Altogether, this study provides a potential mechanism for quadruplex structure formation and resolution during replication and highlights G4s and iMs as endogenous sources of replication stress, which may explain their genomic instability and mutation frequencies in cancer.

molecular biology↗