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Gholami, Z. and Hanley, Q. 
ORCID: 0000-0002-8189-9550,
2014.
Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer.
Bioconjugate Chemistry, 25 (10), pp. 1820-1828.
ISSN 1043-1802
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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 O6-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 hetero-dimers 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.
| Item Type: | Journal article | ||||
|---|---|---|---|---|---|
| Publication Title: | Bioconjugate Chemistry | ||||
| Creators: | Gholami, Z. and Hanley, Q. | ||||
| Publisher: | American Chemical Society | ||||
| Date: | 2014 | ||||
| Volume: | 25 | ||||
| Number: | 10 | ||||
| ISSN: | 1043-1802 | ||||
| Identifiers: |
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| Divisions: | Schools > School of Science and Technology | ||||
| Record created by: | EPrints Services | ||||
| Date Added: | 09 Oct 2015 10:21 | ||||
| Last Modified: | 09 Jun 2017 13:27 | ||||
| URI: | https://irep.ntu.ac.uk/id/eprint/11694 |
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