title: Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer.
abstract: The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks, forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O(6)-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA-directed assembly of homo- and heterodimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template-directed assembly of PNA-modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels, which may find use in the study of assembly processes in cells.
authors.affiliation: School of Science and Technology, Nottingham Trent University , Clifton Lane, Nottingham NG11 8NS, United Kingdom.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Gholami
authors.forename: Quentin
authors.initials: Q
authors.lastname: Hanley
mesh_terms: D004247:DNA / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; C064976:O(6)-benzylguanine; D006147:Guanine; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase