RESUMO
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.
RESUMO
DNA nanotechnology is an emerging field that promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress is the ability to create a nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction that installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled-coil peptide tag. Once installed, the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness and achieving reversible labelling by way of toehold-mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled-coil systems, with epidermal growth factor receptor and endothelin receptor type B, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of epidermal growth factor receptor on CHO cells.
Assuntos
Receptores ErbB/metabolismo , Microscopia de Fluorescência , Ácidos Nucleicos Peptídicos/química , Receptor de Endotelina B/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Receptores ErbB/química , Receptores ErbB/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/metabolismo , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Receptor de Endotelina B/química , Receptor de Endotelina B/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Rapid, site-selective modification of cysteine residues with chloromethyl-triazole derivatives generates pseudo-acyl sLys motifs, mimicking important post-translational modifications. Near-native biotinylation of peptide and protein substrates is shown to be site-selective and modified histone H4 retains functional activity.