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

Area of Science:

• Bioconjugation Chemistry
• Molecular Assembly
• Biophysics

Background:

• Controlled protein alignment is crucial for developing advanced molecular frameworks.
• Existing chemical approaches for protein assembly can be complex and lack orthogonality.
• Peptide nucleic acids (PNAs) offer a promising platform for molecular recognition and assembly.

Purpose of the Study:

• To develop a facile and orthogonal chemical method for controlled protein alignment on DNA scaffolds.
• To create novel protein-PNA conjugates for efficient assembly and analysis.
• To demonstrate the formation of protein dimers and oligomers using DNA-PNA frameworks.

Main Methods:

• Site-selective conjugation of monomeric teal fluorescent protein (mTFP) to PNA via expressed protein ligation (EPL).
• Assembly of mTFP-PNA conjugates on DNA scaffolds to create hetero-FRET systems.
• Characterization using fluorescence intensity, frequency domain lifetime measurements, anisotropy, size exclusion chromatography (SEC), and SDS-PAGE.

Main Results:

• Successful site-selective conjugation of mTFP to PNA.
• Demonstrated Förster Resonance Energy Transfer (FRET) in assembled mTFP-PNA-DNA systems.
• Confirmed the formation of protein dimers and oligomers on extended DNA scaffolds.
• Observed homo-FRET upon assembly of multiple mTFP-PNA constructs.

Conclusions:

• Protein-PNA conjugates are effective components for controlled protein assembly on DNA scaffolds.
• The developed method allows for induced and controllable alignment of proteins.
• This approach enables the creation of sophisticated FRET-based molecular systems and ordered protein structures.