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<p>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 subdivide 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 identifies potential targets for antibiotic development but also establishes a platform for probing the structure and dynamics of RNP assemblies.</p>

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Anti-sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assembly

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月18日

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  • Kai Sheng,
  • Xiyu Dong,
  • Sriram Aiyer,
  • Joan Lee,
  • Selena Đorđević-Marquardt,
  • Dmitry Lyumkis,
  • James R. Williamson

摘要

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 subdivide 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 identifies potential targets for antibiotic development but also establishes a platform for probing the structure and dynamics of RNP assemblies.