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

Z. Gholami, L. Brunsveld, Q. Hanley

Research output: Contribution to journalArticleAcademicpeer-review

13 Citations (Scopus)
1 Downloads (Pure)

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.
Original languageEnglish
Pages (from-to)1378-1386
Number of pages8
JournalBioconjugate Chemistry
Volume24
Issue number8
DOIs
Publication statusPublished - 2013

Fingerprint Dive into the research topics of 'PNA-induced assembly of fluorescent proteins using DNA as a framework'. Together they form a unique fingerprint.

Cite this