Biology subjects

Lestini, R.

Publications and source records attributed to Lestini, R..

3 recordsLinked to original sources

bioRxiv2026

Multi-foci replication domains in Haloferax volcanii visualised by super-resolution microscopy

The archaeon Haloferax volcanii is a unique model organism to investigate replication dynamics and dissect the interplay between multiple origins, alternative replication pathways, and polyploidy. The combination of variable chromosome copy number and asynchronous replication across the cell population gives rise to pronounced phenotypic heterogeneity, further increasing system complexity. To investigate these mechanisms, we implemented (spt-)PALM and STORM microscopy to probe replication dynamics in different cell lines and growth conditions. We established the first STORM-based super-resolution imaging approach of a protein in Haloferax volcanii. Altogether, our results support a model in which each H. volcanii cell replicates a constant fraction of its chromosome copies, irrespective of total ploidy. Consequently, cells with a higher number of chromosomes exhibit proportionally more replication forks, resulting in pronounced ploidy heterogeneity across the population. Moreover, we show that replication foci are not randomly distributed but cluster into discrete Replication Domains containing multiple active sites. This organization persists across different growth conditions and is independent of canonical replication origins, as demonstrated in a strain lacking all four origins. Our findings suggest that replication coordination in H. volcanii is governed by spatial architecture rather than origin-dependent initiation, providing new insights into the evolutionary diversity of replication control mechanisms.

microbiology↗

bioRxiv2026

Highly sensitive enzyme- and amplification-free, quantitative DNA detection using YVO4:Eu luminescent nanoparticle probes

The sensitive detection of nucleic acids is crucial for the accurate diagnosis of infections. In this context, amplification-based methods, such as the quantitative Polymerase Chain Reaction (qPCR) are the gold standard for ultrasensitive DNA or RNA detection and quantification. However, despite its widespread use in developed countries during the COVID-19 pandemic, qPCR remains a costly tool, difficult to implement into low-infrastructure locations. Efforts for the development of alternative tools have yielded high sensitivity approaches but sensitivity is typically reached at the expense of complexity. We here report the development of a simple, sensitive, amplification-, and enzyme-free nucleic acid detection technique using YVO4:Eu luminescent nanoparticles. We established an optimized interaction scheme to efficiently reveal target DNA fragments with nanoparticles. By exploiting the extremely strong absorption of the vanadate matrix in the UV to excite the nanoparticles inducing the characteristic Eu3+ emission at 617 nm via energy transfer, we achieved a highly sensitive (down to 500 particles/mm2; 17,000 particles/well) read-out in standard microplates using a home-made optical reader with light-emitting diode (LED), 275-nm excitation. We reached a 50-aM (30,000 copies/mL) sensitivity for the detection of the 72-base DNA fragment of the SARS-CoV-2 n1 gene. Our new quantitative analytical method detects nucleic acids without amplification with performances close to standard PCR (10,000 copies/mL)1, and could be the basis for a transportable alternative for the diagnosis of infectious diseases.

bioengineering↗

bioRxiv2024

G-quadruplexes in Haloferax volcanii

Archaea, a domain of microorganisms found in diverse environments including the human microbiome, represent the closest known prokaryotic relatives of eukaryotes. This phylogenetic proximity positions them as a relevant model for investigating the evolutionary origins of nucleic acid secondary structures such as G-quadruplexes (G4s), which play regulatory roles in transcription and replication. Although G4s have been extensively studied in eukaryotes, their presence and function in archaea remain poorly characterized. In this study, a genome-wide analysis of the halophilic archaeon Haloferax volcanii identified over 5, 800 potential G4-forming sequences. Biophysical validation confirmed that many of these sequences adopt stable G4 conformations in vitro. Using G4-specific detection tools and super-resolution microscopy, G4 structures were visualized in vivo in both DNA and RNA across multiple growth phases. Comparable findings were observed in the thermophilic archaeon Thermococcus barophilus. Functional analysis using helicase-deficient H. volcanii strains further identified candidate enzymes involved in G4 resolution. These results establish H. volcanii as a tractable archaeal model for G4 biology.

molecular biology↗