INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors

Hör, J.; Jung, J.; Đurica-Mitic, S.; Barquist, L.; Vogel, J.

doi:10.1101/2022.04.11.487859

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bioRxiv · 10.1101/2022.04.11.487859

INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors

Hör, J.·Jung, J.·Đurica-Mitic, S.·Barquist, L.·Vogel, J.

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Abstract

Ribosome profiling (Ribo-seq) is a powerful method for the transcriptome-wide assessment of protein synthesis rates and the study of translational control mechanisms. Yet, Ribo-seq also has limitations. These include difficulties with detection of low abundance transcripts and analysis of translation-modulating molecules such as antibiotics, which are often toxic or challenging to deliver into living cells. Here, we have developed in vitro Ribo-seq (INRI-seq), a cell-free method to analyze the translational landscape of a fully customizable synthetic transcriptome. Using Escherichia coli as an example, we show how INRI-seq can be used to analyze the translation initiation sites of a transcriptome of interest. We also study the global impact of direct translation inhibition by antisense peptide nucleic acid (PNA) to analyze PNA off-target effects. Overall, INRI-seq presents a scalable, sensitive method to study translation initiation in a transcriptome-wide manner without the potentially confounding effects of extracting ribosomes from living cells.

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Hör, J., Jung, J., Đurica-Mitic, S., Barquist, L., Vogel, J.. 2022-04-11. INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors. https://doi.org/10.1101/2022.04.11.487859

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