Orthogonal coiled coils enable rapid covalent labelling of two distinct membrane proteins with peptide nucleic acid barcodes

Abstract

Templated chemistry offers the prospect of addressing specificity challenges occurring in bioconjugation reactions. Here, we show two peptide-templated amide-bond forming reactions that enable the concurrent labelling of two different membrane proteins with two different peptide nucleic acid (PNA) barcodes. The reaction system is based on the mutually selective coiled coil interaction between two thioester-linked PNA-peptide conjugates and two cysteine peptides serving as genetically encoded peptide tags. Orthogonal coiled coil templated covalent labelling is highly specific, quantitative and proceeds within a minute. To demonstrate the usefulness, we evaluated receptor internalisation of two membranous receptors EGFR (epidermal growth factor) and ErbB2 (epidermal growth factor receptor 2) by first staining PNA-tagged proteins with fluorophore-DNA conjugates and then erasing signals from non-internalized receptors via toehold-mediated strand displacement.

Scheme 1. Dual labeling of Cys-Acc-tagged proteins with PNA templated by orthogonal coiled coils.

Fig. 1. (A) Orthogonal coiled coils, PNA-labeling reagents and products formed in coil coil-templated PNA transfer. Fl-UPLC analysis of reactions involving (B) matched and (C) mismatched coiled coil peptides. Conditions: 200 nM Cys-P1-TMR or Cys-P3-TMR, 1200 nM PNA1-P2 or PNA3-P4 in 200 nM phosphate, 1 mM TCEP, 0.1% CHAPS, pH 7.2, 30 °C. (D) One-pot reactions of a Cys-P1-TMR/Cys-P3-C343/PNA1-P2/PNA3-P4-mixture (red traces) overlaid with Fl-UPLC traces for reactions involving Cys-P1-TMR + PNA1-P2 or Cys-P3-C343 + PNA3-P4 in separate pots (black traces). Detector settings: TMR, Ex 550 nm, Em 580 nm; C343, Ex 420 nm, Em 500 nm.

Fig. 2. Dual color live cell labeling. After nuclear staining with Hoechst33342 (blue), (A) Cys-P1-EGFR-eYFP or (B) Cys-P3-ErbB2-eCFP expressing CHO cells were treated with matched (left) or mismatched (right) PNA labeling reagents/fluorophore-DNA. Conditions: 4 min with 100 nM PNA1-P2 (EGFR) or/and PNA3-P4 (ErbB2) in DPBS, washing with HBSS, 4 min hybridization with 200 nM Atto565-DNA1 (EGFR) or Cy7-DNA3 (ErbB2). (C) Simultaneous labeling of Cys-P1-EGFR-eYFP/Cys-P3-EGFR-eCFP cells. Scale bar = 10 μm.

Fig. 3. (A) Dual receptor internalization assay. (B) Fluorescent microscopy imaging of internalized EGFR and ErbB2 in CHO cells. Conditions: see ESI. (C) Quantitative analysis of internalization. Data is presented as internalization relative to the negative control (−) from four independent replicates (n = 100 cells per condition per experiment). Sequences of imager strands and eraser strands are given in 5′ → 3′ direction. Toehold-related sequences are underlined. For other sequences see Table S6-1 (ESI†).