Abstract

(A, B) Scheme of the 4 nanobarcode epitopes (A) and the fluorescent nanobodies used for recognizing them (B). (B) NbRFP-Atto565 in red, NbEGFP-Atto488 in green, NbSyn87-Dylight405 in cyan, NbSyn2-Star635P in magenta. (C) Design of the protein construct VAMP4(1111). Each protein construct contains a target protein (the protein to identify) and a barcode. In this example, the target protein is VAMP4, and its barcode contains the following nonfluorescent epitopes: mCherry (Y71L), GFP (Y66L), syn87, and syn2. The ALFA-tag [10] is present for testing purposes. See S1 Fig for further sequence information. Barcode epitopes recognized by fluorescent nanobodies shown as “ones” in pseudocolors that correspond to the fluorophores used. (D) Nanobarcodes, 15 in total, resulting from a binary combination of 4 nanobarcode-epitopes. Epitopes from left to right: mCherry(Y71L), GFP(Y66L), syn87 and syn2. The nanobody scheme is the same as in (B). (E) The expected cellular protein distribution for the proteins used, according to the literature. (F) Nanobarcode-based identification of the proteins STX6(0011), GFP(0100), and SNAP25(1100). The pseudocolors for merged images correspond to the fluorescence channels of the nanobodies: NbRFP-Atto565 in red, NbGFP-Atto488 in green, NbSyn87-Dylight405 in cyan, and NbSyn2-Star635P in magenta. Scale bar: 20 μm.</p

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