Fig. 1: Genetically encoded affinity reagents (GEARs) function in vivo.

a Overview of genetically encoded probes, their respective sizes, and target epitopes. b Schematic of a binding assay for visualizing GEAR binding in vivo. Nbs or scFvs were fused to EGFP and injected into WT zebrafish embryos, either alone or with tagged versions of nuclear (Nanog) or membrane-bound (Vangl2) targets. Localization of EGFP reflects the in vivo binding ability of these binders. c Single slices of representative microscopy images show EGFP localization when fused to different binders in the absence (top) or presence (middle) of epitope-tagged Nanog. Lines indicate the regions for fluorescence quantification. Line plots (bottom) of normalized EGFP-binder fluorescence intensity in the absence (−target) or presence (+target) of cognate tagged Nanog along lines drawn through cells. Measurements in pixel intervals and centered on the nucleus. d Quantifying and comparing the in vivo binding ability of different GEAR binders to tagged Nanog using the readout of relocalized EGFP fluorescence (nuclear-to-cytoplasmic ratio normalized to a V5-mScarlet-I injection control). Center lines show medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum value in the data. N = 6 (NbMoon) or 5 embryo data points. Each data point is the mean of n = 12 cells per embryo. Data were analyzed using a nonparametric Kruskal–Wallis test, ∗∗∗ p < 0.001 (p-value = 0.00028875). e As in (c) but for GEAR binders in the absence or presence of epitope-tagged Vangl2. f Quantification and comparison of the in vivo binding ability of GEAR binders to tagged Vangl2 using the readout of relocalized EGFP fluorescence as the ratio of membrane-to-cytoplasm, normalized to the V5-mScarlet-I injection control. Center lines show medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum value in the data. N = 4 (NbMoon), 3 (NbVHH05) or 5 embryo data points. Each data point is the mean of n = 12 cells per embryo. Data were analyzed using a nonparametric Kruskal–Wallis test, ∗∗∗ p < 0.001 (p-value = 0.00031782). Scale bars: 10 mm (c, e). See also Figs. S1–3.