Abstract
Proteins and peptides fold into dynamic structures that access a broad functional landscape; however, designing artificial polypeptide systems is still a great challenge. Conversely, DNA engineering is now routinely used to build a wide variety of 2D and 3D nanostructures from hybridization based rules, and their functional diversity can be significantly expanded through site specific incorporation of the appropriate guest molecules. Here we demonstrate a new approach to rationally design 3D nucleic acid–amino acid complexes using peptide nucleic acid (PNA) to assemble peptides inside a 3D DNA nanocage. The PNA-peptides were found to bind to the preassembled DNA nanocage in 5–10 min at room temperature, and assembly could be performed in a stepwise fashion. Biophysical characterization of the DNA-PNA-peptide complex was performed using gel electrophoresis as well as steady state and time-resolved fluorescence spectroscopy. Based on these results we have developed a model for the arrangement of the PNA-peptides inside the DNA nanocage. This work demonstrates a flexible new approach to leverage rationally designed nucleic acid (DNA-PNA) nanoscaffolds to guide polypeptide engineering.
Materials and Methods section, calculations for nucleobase and dye extinction coefficients, predicted and calculated DNA-PNA dissociation temperatures, evaluation of spectroscopic data including calculating quantum yields, fluorescence lifetimes, anisotropies, FRET efficiencies, Förster radii, and distances; supporting results and discussion on the DNA-PNA-peptide complex design and PNA-peptide synthesis; Figure S1, DNA and PNA sequences and schematic showing their arrangement in the complex; Figure S2, complex nomenclature with list of constituent DNA and PNA-peptide strand names; Figure S3, MALDI mass spectrum of the synthesized PNA-peptides; Figure S4, RP-HPLC chromatograms of the synthesized PNA-peptides; Figure S5, SE-HPLC chromatograms of the DNA nanocage with and without PNA-peptides; Figure S6, native PAGE of the DNA nanocage incubated with PNA1-GPG at 4 °C; Figure S7, chemical structures of FAM and TMR; Figure S8, absorbance of the labeled PNA1-peptide hybridized to the DNA nanocage; Figure S9, temperature dependence of FAM fluorescence; Figure S10, time course of PNA1-peptide hybridization with the DNA nanocage; Table S1, lifetime components and calculated average lifetimes of the donor and acceptor. This material is available free of charge via the Internet at http://pubs.acs.org.
