Low temperature assembly of functional 3D DNA-PNA-protein complexes

Area of Science:

• Biochemistry
• Nanotechnology
• Molecular Biology

Background:

• Investigating protein interactions within biological systems is crucial but challenging.
• DNA nanostructures offer a platform for organizing molecules, but flexible protein arrangement in 3D is needed.
• Peptide nucleic acid (PNA) can bridge the gap between DNA nanostructures and proteins.

Purpose of the Study:

• To develop a flexible method for assembling functional proteins into 3D DNA nanocages using PNA.
• To investigate the influence of protein surface charge on its interaction with DNA nanocages.
• To maintain protein function and explore engineered protein complexes.

Main Methods:

• Utilized peptide nucleic acid (PNA) for protein conjugation and assembly into 3D DNA nanocages.
• Employed toehold-mediated DNA strand displacement for purifying PNA-protein conjugates.
• Applied gel electrophoresis and fluorescence spectroscopy to analyze protein-DNA interactions.

Main Results:

• Successfully assembled cytochrome c and azurin proteins into separate 3D DNA nanocages, preserving protein function.
• Demonstrated rapid assembly of PNA-proteins within DNA nanocages at room temperature and 11 °C.
• Modeled interactions showing charge-dependent behavior: positively charged cytochrome c interacts with the DNA nanocage, while negatively charged azurin is repelled.

Conclusions:

• PNA-DNA nanostructures provide a flexible approach for controlled assembly of functional proteins.
• This method facilitates the study of protein interactions and the engineering of complex 3D protein architectures.
• The findings open avenues for creating novel protein-based nanomaterials and studying protein behavior in controlled environments.