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

Proteins have evolved to carry out nearly all the work required of living organisms within complex inter- and intracellular environments. However, systematically investigating the range of interactions experienced by a protein that influence its function remains challenging. DNA nanostructures are emerging as a convenient method to arrange a broad range of guest molecules. However, flexible methods are needed for arranging proteins in more biologically relevant 3D geometries under mild conditions that preserve protein function. Here we demonstrate how peptide nucleic acid (PNA) can be used to control the assembly of cytochrome c (12.5 kDa, pI 10.5) and azurin (13.9 kDa, pI 5.7) proteins into separate 3D DNA nanocages, in a process that maintains protein function. Toehold-mediated DNA strand displacement is introduced as a method to purify PNA-protein conjugates. The PNA-proteins were assembled within 2 min at room temperature and within 4 min at 11 °C, and hybridize with even greater efficiency than PNA conjugated to a short peptide. Gel electrophoresis and steady state and time-resolved fluorescence spectroscopy were used to investigate the effect of protein surface charge on its interaction with the negatively charged DNA nanocage. These data were used to generate a model of the DNA-PNA-protein complexes that show the negatively charged azurin protein repelled away from the DNA nanocage while the positively charged cytochrome c protein remains within and closely interacts with the DNA nanocage. When conjugated to PNA and incorporated into the DNA nanocage, the cytochrome c secondary structure and catalytic activity were maintained, and its redox potential was reduced modestly by 20 mV possibly due to neutralization of some positive surface charges. This work demonstrates a flexible new approach for using 3D nucleic acid (PNA-DNA) nanostructures to control the assembly of functional proteins, and facilitates further investigation of protein interactions as well as engineer more elaborate 3D protein complexes.