Anti-sense oligonucleotide inhibited iSAT reaction

ABSTRACT: Investigating the intricate and rapid folding kinetics of large RNA-protein complexes (RNPs), like the bacterial ribosome, remains a formidable challenge in structural biology. Previous genetic approaches to probe assembly have focused on modulating the expression of either r-proteins or assembly factors. Here, anti-sense oligonucleotides (ASOs) were used to disrupt native RNA/RNA and RNA/protein interactions, in order to generate previously uncharacterized folding intermediates. In an in vitro co-transcriptional ribosome assembly assay, 10 assembly inhibitor ASOs were identified. Using cryo-electron microscopy, 38 intermediate structures were determined resulting from the specific inhibitions generated by 6 inhibitory ASOs. A notable intermediate class provided compelling evidence for independent rRNA domain folding before proper interdomain docking. Three PNAs targeting domain-I of 23S rRNA further subdivided the previously identified assembly core into smaller blocks representing the earliest steps in assembly. The resulting assembly graph reveals template-directed RNA docking of defined regions as foldons, and domain consolidation, which provides a hierarchical view of the RNP assembly process. This approach not only identified potential targets for antibiotic development but also established a platform for probing the structure and dynamics of RNP assemblies.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Escherichia Coli

SUBMITTER: Kai Sheng  

LAB HEAD: James Williamson

PROVIDER: PXD064127 | Pride | 2025-07-28

REPOSITORIES: Pride