Single-molecule FRET: sRNA·Hfq binding to mRNA, imaged in real time

01

TIRF · smFRET · kinetics

Single-molecule TIRF microscopy

Significant effects of sRNAs in gene expression are observed as quickly as 1–2 minutes from signal induction, yet we do not fully understand how the coordination between these processes occurs so efficiently on this time scale. To address this, we monitor the assembly of RNA–protein complexes as it evolves in real time.

To dive deep into the molecular mechanism of fast biological processes, we need a method that:

  • provides high temporal resolution, from milliseconds to minutes;
  • resolves conformational changes in RNA–protein or RNA–RNA complexes;
  • allows visualisation of the various pathways a reaction can take.

Single-molecule TIRF microscopy lets us visualise hundreds of biomolecules immobilised on a microscope slide, each analysed separately. Molecules are fluorescently labelled and interactions detected by colocalisation of fluorophores or FRET.

Assembly of sRNA, Hfq and the degradosome on mRNA, leading to RNA degradation

02

RNA · Hfq · degradosome

Post-transcriptional regulation of gene expression in bacteria

Our lab investigates the coordination between RNA targeting, degradation, and translation in bacteria. Many bacteria are beneficial or harmless, but some cause infectious diseases, often regulated by the RNA chaperone protein Hfq and small RNAs (sRNAs). Hfq facilitates base pairing between sRNAs and target mRNAs, leading to changes in translation initiation or RNA degradation.

RNA degradation is carried out by the degradosome, a complex of RNase E, enolase, RhlB helicase, and PNPase. The degradosome can preassemble with sRNA and Hfq, potentially enabling more efficient regulation of mRNA. We study how alternative assembly pathways of these complexes influence regulatory outcomes.

Additionally, we aim to understand how these complexes interact with mRNA during translation. By reconstituting active translation in vitro, we investigate the real-time coordination of RNA targeting and degradation.

Phage and host genomes inside an infected cell, and the RNAs each produces

03

Phages · CRAB · proteomics

Phage–host interactions

Antibiotic resistance, driven by bacterial adaptation, poses a significant challenge, particularly with ESKAPE pathogens such as carbapenem-resistant Acinetobacter baumannii (CRAB). Bacteriophages, viruses that infect and lyse bacterial cells, offer a promising alternative to traditional antibiotics.

Our research focuses on mapping interactions between phages and bacterial hosts across multiple biological levels to identify phage factors with antimicrobial potential. We are studying Acinetobacter and Escherichia phages using transcriptomic and proteomic analyses, as well as biochemical and single-molecule approaches.