Professor Achillefs Kapanidis

Abstract:

The increasing frequency and magnitude of viral outbreaks in recent decades, epitomized by the COVID-19 pandemic, has resulted in an urgent need for rapid and sensitive diagnostic methods. Here, we present a methodology for virus detection and identification that uses a convolutional neural network to distinguish between microscopy images of fluorescently labeled intact particles of different viruses. Our assay achieves labeling, imaging, and virus identification in less than 5 min and does not require any lysis, purification, or amplification steps. The trained neural network was able to differentiate SARS-CoV-2 from negative clinical samples, as well as from other common respiratory pathogens such as influenza and seasonal human coronaviruses. We were also able to differentiate closely related strains of influenza, as well as SARS-CoV-2 variants. Additional and novel pathogens can easily be incorporated into the test through software updates, offering the potential to rapidly utilize the technology in future infectious disease outbreaks or pandemics. Single-particle imaging combined with deep learning therefore offers a promising alternative to traditional viral diagnostic and genomic sequencing methods and has the potential for significant impact.

Abstract:

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)-σ⁷⁰ holoenzyme to the promoter DNA. This initial RNAP-promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; these changes lead to the catalytically active open complex (RP_O) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RP_O and putative intermediates on its formation pathway. Here, we employ single-molecule fluorescence microscopy to interrogate the conformational dynamics and reaction kinetics during real-time RP_O formation on a consensus lac promoter. We find that the promoter opening may proceed rapidly from the closed to open conformation in a single apparent step, or may instead involve a significant intermediate between these states. The formed RP_O complexes are also different with respect to their transcription bubble stability. The RNAP cleft loops, and especially the β' rudder, stabilise the transcription bubble. The RNAP interactions with the promoter upstream sequence (beyond -35) stimulate transcription bubble nucleation and tune the reaction path towards stable forms of the RP_O.