PNA-induced assembly of fluorescent proteins using DNA as a framework

GHOLAMI, Z., BRUNSWELD, L. and HANLEY, Q., 2013. PNA-induced assembly of fluorescent proteins using DNA as a framework. Bioconjugate Chemistry, 24 (8), pp. 1378-1386. ISSN 1043-1802

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Abstract

Controlled alignment of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this work, protein−PNA conjugates are brought forward as new chemical components allowing efficient assembly and alignment on DNA scaffolds. Site-selective monomeric teal fluorescent protein (mTFP)−peptide nucleic acid (PNA) (mTFP-PNA) conjugation was achieved by covalent linkage of the PNA to the protein through expressed protein ligation (EPL). A DNA beacon, with 6-Fam and Dabcyl at its ends, acts as a framework to create an assembled hetero-FRET system with the mTFP-PNA conjugate. Using fluorescence intensity, frequency domain lifetime measurements, and anisotropy measurements, the system was shown to produce FRET as indicated by decreased donor intensity, decreased donor lifetime, and increased donor anisotropy. Extension of the DNA scaffold allowed for the assembly of multiple mTFP-PNA constructs. Efficient formation of protein dimers and oligomers on the DNA-PNA frameworks could be shown, as visualized via size exclusion chromatography (SEC) and electrophoresis (SDS-PAGE). Assembly of multiple proteins in a row induced homo-FRET for the mTFP-PNA’s assembled on the DNA scaffolds. The oligonucleotide framework allows an induced and controllable assembly of proteins by fusing them to PNAs directed to align on DNA scaffolds.

Item Type: Journal article
Publication Title: Bioconjugate Chemistry
Creators: Gholami, Z., Brunsweld, L. and Hanley, Q.
Publisher: American Chemical Society
Date: 2013
Volume: 24
Number: 8
ISSN: 1043-1802
Identifiers:
NumberType
10.1021/bc400202sDOI
Rights: Copyright © 2013 American Chemical Society
Divisions: Schools > School of Science and Technology
Depositing User: EPrints Services
Date Added: 09 Oct 2015 10:30
Last Modified: 09 Jun 2017 13:31
URI: http://irep.ntu.ac.uk/id/eprint/14021

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