Molecular Horizons Seminar: Publication of the Year 2024 Winners

Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein

Nick Marzano

Pentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be modified to bind to a specific ssRNA nucleobase. As such, there is interest in developing ‘designer’ PPRs (dPPRs) for a range of biotechnology applications, including diagnostics or in vivo localisation of ssRNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. To address this, we determined the structure of a dPPR bound to its target sequence and used two- and three-colour single-molecule FRET to interrogate the mechanism of ssRNA binding to individual dPPRs in real time.

Deep Characterisation of the sn-Isomer Lipidome Using High-Throughput Data-Independent Acquisition and Ozone-Induced Dissociation

Jesse Michael

Mass Spectrometry-based lipidomics datasets regularly annotate lipid species to only a limited level of structural detail (e.g. phosphatidylcholine PC 34:1), masking a great deal of structural diversity crucial to understanding biological origin and function. Here, we report the extension of “CID/OzID” to a DIA shotgun lipidomics workflow for the deep characterisation of lipid sn-isomers. We measured 966 distinct PC and phosphatidylethanolamine species across several cell lines, representing an order of magnitude increase in the number of isomer-resolved molecular lipid species compared to previous reports and revealing that sn-isomer populations are tightly regulated and vary greatly across biological systems. The sensitivity of this method is further demonstrated by the identification of a range of novel PCs with unusual ultra-long monounsaturated acyl chains at the sn-1 position.