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

Artificial DNA, PNA & XNA
Justin D FloryPetra Fromme

Abstract

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...Continue Reading

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Citations

Mar 19, 2016·Chembiochem : a European Journal of Chemical Biology·Alexander Iain TaylorPhilipp Holliger
Dec 18, 2018·ACS Omega·Pramod M SabaleSeergazhi G Srivatsan

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Methods Mentioned

BETA
biosensor
fluorescence spectroscopy

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