TY - JOUR
T1 - Purification and assembly of thermostable Cy5 labeled γ-PNAs into a 3D DNA nanocage
AU - Flory, Justin D.
AU - Johnson, Trey
AU - Simmons, Chad R.
AU - Lin, Su
AU - Ghirlanda, Giovanna
AU - Fromme, Petra
N1 - Funding Information:
This work was supported by the Center for Bio-Inspired Solar Fuel Production, an Energy Frontier Research Center funded by the US. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0001016.
PY - 2014
Y1 - 2014
N2 - PNA is hybrid molecule ideally suited for bridging the functional landscape of polypeptides with the structural diversity that can be engineered with DNA nanostructures. However, PNA can be more challenging to work with in aqueous solvents due to its hydrophobic nature. A solution phase method using strain promoted, copper free click chemistry was developed to conjugate the fluorescent dye Cy5 to 2 bifunctional PNA strands as a first step toward building cyclic PNA-polypeptides that can be arranged within 3D DNA nanoscaffolds. A 3D DNA nanocage was designed with binding sites for the 2 fluorescently labeled PNA strands in close proximity to mimic protein active sites. Denaturing polyacrylamide gel electrophoresis (PAGE) is introduced as an efficient method for purifying charged, dye-labeled PNA conjugates from large excesses of unreacted dye and unreacted, neutral PNA. Elution from the gel in water was monitored by fluorescence and found to be more efficient for the more soluble PNA strand. Native PAGE shows that both PNA strands hybridize to their intended binding sites within the DNA nanocage. Förster resonance energy transfer (FRET) with a Cy3 labeled DNA nanocage was used to determine the dissociation temperature of one PNA-Cy5 conjugate to be near 50°C. Steady-state and time resolved fluorescence was used to investigate the dye orientation and interactions within the various complexes. Bifunctional, thermostable PNA molecules are intriguing candidates for controlling the assembly and orientation of peptides within small DNA nanocages for mimicking protein catalytic sites.
AB - PNA is hybrid molecule ideally suited for bridging the functional landscape of polypeptides with the structural diversity that can be engineered with DNA nanostructures. However, PNA can be more challenging to work with in aqueous solvents due to its hydrophobic nature. A solution phase method using strain promoted, copper free click chemistry was developed to conjugate the fluorescent dye Cy5 to 2 bifunctional PNA strands as a first step toward building cyclic PNA-polypeptides that can be arranged within 3D DNA nanoscaffolds. A 3D DNA nanocage was designed with binding sites for the 2 fluorescently labeled PNA strands in close proximity to mimic protein active sites. Denaturing polyacrylamide gel electrophoresis (PAGE) is introduced as an efficient method for purifying charged, dye-labeled PNA conjugates from large excesses of unreacted dye and unreacted, neutral PNA. Elution from the gel in water was monitored by fluorescence and found to be more efficient for the more soluble PNA strand. Native PAGE shows that both PNA strands hybridize to their intended binding sites within the DNA nanocage. Förster resonance energy transfer (FRET) with a Cy3 labeled DNA nanocage was used to determine the dissociation temperature of one PNA-Cy5 conjugate to be near 50°C. Steady-state and time resolved fluorescence was used to investigate the dye orientation and interactions within the various complexes. Bifunctional, thermostable PNA molecules are intriguing candidates for controlling the assembly and orientation of peptides within small DNA nanocages for mimicking protein catalytic sites.
KW - DBCO, dibenzocyclooctyl
KW - DNA nanotechnology
KW - DTNB, 5, 5′-dithiobis-(2-nitrobenzoic acid)
KW - EtBr, ethidium bromide
KW - IEX-FPLC, ion-exchange fast protein liquid chromatography
KW - MALDI-MS, matrix assisted laser desorption ionization mass spectrometry
KW - PAGE, polyacrylamide gel electrophoresis
KW - PNA, peptide nucleic acid
KW - RP-HPLC, reverse-phase high pressure liquid chromatography
KW - TCEP, tris(2-carboxyethyl)phosphine
KW - biomimicry
KW - copper-free click chemistry
KW - fluorescence
KW - self-assembly
KW - γ-PNA
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U2 - 10.4161/1949095X.2014.992181
DO - 10.4161/1949095X.2014.992181
M3 - Article
C2 - 25760314
AN - SCOPUS:84944146341
SN - 1949-095X
VL - 5
SP - 1
EP - 8
JO - Artificial DNA, PNA & XNA
JF - Artificial DNA, PNA & XNA
IS - 3
ER -