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

Justin D. Flory, Trey Johnson, Chad R. Simmons, Su Lin, Giovanna Ghirlanda, Petra Fromme

Research output: Contribution to journalArticle

2 Citations (Scopus)

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-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.

Original languageEnglish
Pages (from-to)1-8
Number of pages8
JournalArtificial DNA, PNA & XNA
Volume5
Issue number3
DOIs
Publication statusPublished - 2014

Fingerprint

Purification
DNA
Coloring Agents
Electrophoresis
Peptides
Catalytic Domain
Fluorescence
Binding Sites
Click Chemistry
Native Polyacrylamide Gel Electrophoresis
Molecules
Nanostructures
Energy Transfer
Fluorescent Dyes
Energy transfer
cyanine dye 5
Copper
Polyacrylamide Gel Electrophoresis
Proteins
Gels

Keywords

  • biomimicry
  • copper-free click chemistry
  • DBCO, dibenzocyclooctyl
  • DNA nanotechnology
  • DTNB, 5, 5′-dithiobis-(2-nitrobenzoic acid)
  • EtBr, ethidium bromide
  • fluorescence
  • IEX-FPLC, ion-exchange fast protein liquid chromatography
  • MALDI-MS, matrix assisted laser desorption ionization mass spectrometry
  • PAGE, polyacrylamide gel electrophoresis
  • PNA, peptide nucleic acid
  • RP-HPLC, reverse-phase high pressure liquid chromatography
  • self-assembly
  • TCEP, tris(2-carboxyethyl)phosphine
  • γ-PNA

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Purification and assembly of thermostable Cy5 labeled γ-PNAs into a 3D DNA nanocage. / Flory, Justin D.; Johnson, Trey; Simmons, Chad R.; Lin, Su; Ghirlanda, Giovanna; Fromme, Petra.

In: Artificial DNA, PNA & XNA, Vol. 5, No. 3, 2014, p. 1-8.

Research output: Contribution to journalArticle

Flory, Justin D. ; Johnson, Trey ; Simmons, Chad R. ; Lin, Su ; Ghirlanda, Giovanna ; Fromme, Petra. / Purification and assembly of thermostable Cy5 labeled γ-PNAs into a 3D DNA nanocage. In: Artificial DNA, PNA & XNA. 2014 ; Vol. 5, No. 3. pp. 1-8.
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AU - Fromme, Petra

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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.

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KW - PAGE, polyacrylamide gel electrophoresis

KW - PNA, peptide nucleic acid

KW - RP-HPLC, reverse-phase high pressure liquid chromatography

KW - self-assembly

KW - TCEP, tris(2-carboxyethyl)phosphine

KW - γ-PNA

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