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. 2022 Jul 15;12:12126. doi: 10.1038/s41598-022-15526-7

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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iCodon generates fluorescent variants with desired expression levels. (A) Illustration of 12 GFP variants generated by iCodon differing only in synonymous mutations and its predicted mRNA stability. All GFP variants were transfected in 293T cells and the fluorescence was measured by cytometry analysis. (B) Scatter plot showing a positive correlation between predicted mRNA stability and GFP fluorescence intensity in 293T transfected cells (r = 0.89, p < 2.2 × 10^–16, Pearson correlation test). n = 12 for all tested variants. (C) Barplot displaying the number of codon (gray) or nucleotide (purple) changes in all 12 GFP variants compared to EGFP. (D) Four GFP variants were co-injected with TagRFP into 1-cell stage zebrafish embryos and imaged 24 h post injection (hpi). The mRNA stability predictions by iCodon in zebrafish are indicated in brackets. iCodon predictions are slightly different for zebrafish compared to humans as codon optimality is not identical between these two species. (E) Microscopy images of injected zebrafish embryos after 24 hpi. Scale bars represent 200 µm. (F) Quantification of the differences of fluorescence intensity of GFP relativized by RFP fluorescence from injected zebrafish embryos. p values and replicates (n) are indicated.