PNA-protein conjugates for nano-scale modeling of protein aggregates
Abstract Programmable assembly of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this research, the unique characteristics of PNA were applied to create controllable protein assemblies directed by precise PNA-DNA hybridization. The signatures of assembly were studied via FRET, providing a powerful tool which should be effective in live system imaging. Two model systems were developed in this study. In the first model system, site-selective conjugation of monomeric teal fluorescent protein (mTFP) to PNA was achieved by covalent linkage of mTFP to PNA via expressed protein ligation. The mTFP-PNA conjugates were efficiently aligned on a DNA beacon, to create a hetero-FRET system. The FRET indicated by decrease of fluorescence intensity and lifetime of the donor and an increase of donor anisotropy. The assembly of similar multiple mTFP-PNA constructs on DNA scaffolds provided dimeric and oligomeric forms which were studied by SEC-HPLC and SDS-PAGE. A decrease of anisotropy was exhibited due to homo-FRET following induced formation of dimers and oligomers. In the second model system, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers and oligomers.
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