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Nanopore microscope identifies RNA isoforms with structural colours

Abstract

Identifying RNA transcript isoforms requires intricate protocols that suffer from various enzymatic biases. Here we design three-dimensional molecular constructs that enable identification of transcript isoforms at the single-molecule level using solid-state nanopore microscopy. We refold target RNA into RNA identifiers with designed sets of complementary DNA strands. Each reshaped molecule carries a unique sequence of structural (pseudo)colours. Structural colours consist of DNA structures, protein labels, native RNA structures or a combination of all three. The sequence of structural colours of RNA identifiers enables simultaneous identification and relative quantification of multiple RNA targets without prior amplification. Our Amplification-free RNA TargEt Multiplex Isoform Sensing (ARTEMIS) method reveals structural arrangements in native transcripts in agreement with published variants. ARTEMIS discriminates circular and linear transcript isoforms in a one-step, enzyme-free reaction in a complex human transcriptome using single-molecule read-out.

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Fig. 1: ARTEMIS identifies multiple RNA targets using structural colours and nanopore microscopy.
Fig. 2: One pot reaction for multiplexed amplification-free identification of 18S rRNA, 28S rRNA and MS2 RNA.
Fig. 3: ARTEMIS discriminates engineered, alternative splicing isoforms resulting from any physical transcript arrangement.
Fig. 4: Structural colours by programmable RNA origami self-assembly.
Fig. 5: ARTEMIS discriminates alternative splicing isoforms of messenger RNA and long non-coding RNA in a complex human transcriptome mixture.

Data availability

Data supporting the findings of this study are available in the main text and the Supplementary Information. Additional raw data are available at https://doi.org/10.17863/CAM.87123. Source data are provided with this paper.

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Acknowledgements

We thank J. Zhu and M. Fletcher for the critical reading of the manuscript and useful suggestions. We thank the Howarth Lab from the University of Oxford for the monovalent streptavidin. U.F.K. acknowledges funding from a European Research Council Consolidator grant (DesignerPores no. 647144) and European Research Council Proof-Of-Concept grant (PoreDetect no. 899538). F.B. acknowledges funding from George and Lilian Schiff Foundation Studentship, the Winton Programme for the Physics of Sustainability Ph.D. Scholarship and St John’s College Benefactors’ Scholarship.

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Authors and Affiliations

Authors

Contributions

F.B. conceived the idea. F.B. and U.F.K. designed the study. F.B. performed the experiments and analysed the data. F.B. and U.F.K. wrote the manuscript.

Corresponding author

Correspondence to Ulrich Felix Keyser.

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Competing interests

F.B. and U.F.K. are inventors for the ARTEMIS method (United Kingdom patent application no. 2113935.7, in process) submitted by Cambridge Enterprise on the behalf of the University of Cambridge. U.F.K. is a cofounder of Cambridge Nucleomics.

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Nature Chemistry thanks Sergii Pud, Adam Hall and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Figs. 1–24, Tables 1–16, Materials and Methods, and text.

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Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data and RNA structure prediction files.

Source Data Fig. 5

Statistical source data.

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Bošković, F., Keyser, U.F. Nanopore microscope identifies RNA isoforms with structural colours. Nat. Chem. (2022). https://doi.org/10.1038/s41557-022-01037-5

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