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. 2024 Sep 5;52(18):10754–10774. doi: 10.1093/nar/gkae741

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© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — RNA-seq analysis of on-target and off-target effects of siVIM-270 with 2′-formamido modifications (A-C). MA plots showing log₂ fold changes (vertical axis) and average log₁₀ transcripts per million (TPM) (horizontal axis) of the expression levels of transcripts from cells transfected with siVIM-270 and its variants versus those from mock-transfected cells. Dark blue dots represent 676 transcripts with the siVIM-270 SM sequence (off-target transcripts) in their 3′ UTRs, while light blue dots represent 8076 other transcripts without the SM sequence (not off-target transcripts). (D-F) Cumulative distributions of transcripts showing log₂ fold changes (horizontal axis) and cumulative fractions (vertical axis). Red and black lines show the results for off-target and non-off-target transcripts, respectively. (G) VIM mRNA levels calculated by TPM compared to mock. (H) Seed-dependent off-target effects shown by mean log₂ fold change for unmodified siVIM-270 and 2′-formamido-modified siVIM-270s at positions 3 or 8. Statistical significance for each group was determined by comparing the cumulative distribution differences of siRNA SM transcripts using the Wilcoxon rank-sum test (**P < 0.01, ***P < 0.001).