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

Abstract

Abbreviations: PNA, peptide nucleic acid; DBCO, dibenzocyclooctyl; PAGE, polyacrylamide gel electrophoresis; RP-HPLC, reversephase high pressure liquid chromatography; MALDI-MS, matrix assisted laser desorption ionization mass spectrometry; IEX-FPLC, ion-exchange fast protein liquid chromatography; EtBr, ethidium bromide; DTNB, 5, 5 0 -dithiobis-(2-nitrobenzoic acid); TCEP, tris(2-carboxyethyl)phosphine.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, dyelabeled 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. F€ orster 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.

Nanostructures from Synthetic Genetic Polymers

“…[39] However, de novo design in the absence of detailed knowledge on XNA structural and conformational parameters is challenging. Hybrid nanostructures based on DNA designs have previously been demonstrated to retain overall architecture, despite invasion by strands composed of, inter alia, peptiden ucleic acids (PNA) [41][42][43] or phosphorothioate DNA (PS-DNA). [32] Furthermore, af unctional Phi29 DNA-packing motor can be assembled with partial substitution of RNA components with 2'-fluro-2'-deoxy-ribofuranose nucleic acid (2'F-RNA).…”

Perspectives on conformationally constrained peptide nucleic acid (PNA): insights into the structural design, properties and applications

“…Short bifunctional gPNA probes with a thiol group on the backbone could be site-specifically and stably attached to the DNA tetrahedron which could be used for programmed assembly of ligands or biomolecules. 236 In the last example, the control of the supramolecular architecture of amphiphilic PNA was described by Heemstra et al (Fig. 42).…”

“…Short bifunctional γPNA probes with a thiol group on the backbone could be site-specifically and stably attached to the DNA tetrahedron which could be used for programmed assembly of ligands or biomolecules. 236 …”

Clickable PNA Probes for Imaging Human Telomeres and Poly(A) RNAs

“…Several chemoselective reactions, including copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC), strain-promoted azide–alkyne cycloaddition (SPAAC), thiol–ene coupling, azide–phosphine Staudinger ligation, inverse electron demand Diels–Alder, and palladium-catalyzed reactions have been described for labeling biomolecules. − In particular, azide–alkyne cycloaddition reaction has gained prominence as it is fast, highly chemoselective, and bioorthogonal, which has enabled its use in cell-free as well as in cellular systems. Initially, this bioconjugation method was performed on a solid support to prepare labeled PNA oligomers. ,− Subsequently, solution-phase click labeling was conceived, wherein PNAs conjugated to cell-penetrating peptides, mesoporous silica nanoparticles, cross-linking agents, and fluorophores were prepared. − However, given the usefulness of PNA in molecular diagnosis, establishment of chemical labeling strategies, which will provide direct access to a repertoire of PNA probes in a modular fashion, is constantly needed.…”