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.
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.
A pair of orthogonal coiled coils templates highly specific live cell bioconjugation of two different proteins. PNA tagging and hybridisation with fluorophore-DNA reporters enables rapid dual receptor internalisation analysis of EGFR and ErbB2.
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.
Synthetic routes to drug products typically introduce several potentially mutagenic impurities (PMIs) which require controlling to a safe level in the final drug substance, generally directed by the ...control options within the ICH M7 guideline. These impurities are most commonly introduced due to their specific synthetic utility; however, the formation of a PMI can also occur indirectly from a combination of otherwise nonmutagenic sources, as was the case for NDMA within valsartan. Identifying these formation risks currently relies on manually assessing the synthetic route, a process requiring extensive knowledge and potentially liable to oversight. Herein we report on the development of functionality within an in silico risk assessment tool to facilitate the identification of synthetic stages which introduce the risk of formation of PMIs for industrial and/or regulatory users.
Synthetic routes to drug substances can result in the introduction of potentially mutagenic impurities (PMIs). The ICH M7 guideline offers a range of control options that assures that the level of ...this impurity in the drug substance and drug product is below the acceptable limit. Control option 4 leverages the use of predicted purge and outlines the method of utilizing chemical knowledge, literature evidence, and process knowledge to predict the purge of a PMI during drug substance synthesis. If the predicted levels of an impurity in the API are sufficiently lower than the acceptable limit, there will be no requirement for routine analytical testing. Mirabilis is an in silico tool that offers a standardized and conservative approach for the purge calculation of PMIs. The recent developments to methodology for assessing reactivity-based purges within Mirabilis aim to make predictions in a manner more consistent with how a chemist would assess the same situation. The condition-based approach considers the reactants and reagents present and how they may interact, bringing about significant improvements to the specificity and applicability of predictions versus the previous transformation-based approach. Purge predictions for reactivity are now available for 30 impurity types, including N-nitroso compounds and secondary aliphatic amines. Three case studies demonstrate how the new approach provides purge calculations that better align with expert users due to the increased specificity of the predictions.
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.
Chloromethyl triazoles are shown to be cysteine selective alkylation reagents for 'near-native' post-translational modification of protein and peptide substrates.
Chloromethyl triazoles are shown to be cysteine selective alkylation reagents for ‘near-native’ post-translational modification of protein and peptide substrates.
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.
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