Low Temperature Assembly of Functional 3D DNA-PNA-Protein Complexes

Abstract

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.

Purification and assembly of thermostable Cy5 labeled γ-PNAs into a 3D DNA nanocage

“…The change in Cy3 fluorescence intensity as a function of temperature is plotted in Figure 4B and fit with a sigmoidal dose response curve, where the inflection point indicates the dissociation temperature (T D ). The determined T D for PNA3-Cy5 (47.3 C) is in agreement with theory 28 (48.9 C) and confirms our earlier hypothesis that increasing the purine content could be used to increase the thermal stability of our previous DNA-PNA-peptide 22 and DNA-PNA-protein 23 complexes without increasing the PNA sequence length, which may affect the DNA nanocage assembly.…”

“…DNA-PNA complex design Previously we incorporated 2 fluorescently labeled peptides 22 or proteins 23 within a 3D DNA nanocage using PNA linkers. Briefly, we modified an existing DNA tetrahedron design by introducing 8nt single stranded domains complementary to a PNA strand functionalized on the N terminus with a short peptide or small protein oriented toward the center of the DNA nanocage.…”

“…The Cy5 labeled g-PNAs were assembled into DNA nanocages under benign conditions that were previously shown to preserve polypeptide function. 23 Energy transfer between PNA3-Cy5 and DNA1-Cy3 was used to confirm the high thermal stability of the PNA sequence. Interestingly, no energy transfer is observed for PNA1-Cy5 that may be due to the high anisotropy of Cy3 in that complex keeping the dyes at orthogonal orientations.…”

“…18,19 PNA has also been assembled within DNA scaffolds to investigate applications in bionanotechnology. [20][21][22] Previously we showed the versatility of using PNA linkers for assembling peptides 22 and proteins 23 into a 3D DNA nanocage. The assembly process was rapid and could be performed at or below room temperature, providing mild conditions that preserved the function of the attached polypeptides.…”

Chemistry Can Make Strict and Fuzzy Controls for Bio-Systems: DNA Nanoarchitectonics and Cell-Macromolecular Nanoarchitectonics

“…Furthermore, cytochrome c (12.5 kDa) and azurin (13.9 kDa) were bound to 3D DNA nanocages by using a 8-mer PNA linker. 254 The positively charged cytochrome c (pI 10.5) remained within the DNA nanocage and closely interacted with this negatively charged cage. Its secondary structure and catalytic activity cytochrome c were not much altered there.…”