Abstract
Fluorescence anisotropy may be used as an indicator of homo-FRET making it an attractive reported of oligomer formation and aggregation. A model system was created consisting of a monomeric teal fluorescent protein peptide nucleic acid (mTFP-PNA) fusion. The mTFP-PNA monomer units produced by expressed protein ligation (EPL). An mTFP with a C-terminal thioester group was expressed with a modified intein and subsequently conjugated to a PNA with an N-terminal thiol group. This generates a native peptide bond at the ligation site. Addition of a complementary template DNA to solutions of mTFP-PNA monomer units resulted in template directed induced assembly. Assembly was confirmed by SDS-PAGE, mass spectrometry, and size exclusion HPLC.Fluorescence anisotropy was monitored over the course of a mTFP-PNA:DNA titration. Template coding for dimer formation was studied. The anisotropy showed a decreasing trend related to homo-FRET in the assembled forms. A maximal reduction (41%) was observed at a DNA to mTFP-PNA ratio of 1:2. Anisotropy, then increased steadily up to a 1:1 ratio.This study demonstrates an inducibly assembled homo-FRET system using expressed protein ligation which may be extended to study oligomerization and cluster formation in living systems.
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