Programmable multivalent display of receptor ligands using peptide nucleic acid nanoscaffolds

Abstract

Multivalent effects dictate the binding affinity of multiple ligands on one molecular entity to receptors. Integrins are receptors that mediate cell attachment through multivalent binding to peptide sequences within the extracellular matrix, and overexpression promotes the metastasis of some cancers. Multivalent display of integrin antagonists enhances their efficacy, but current scaffolds have limited ranges and precision for the display of ligands. Here we present an approach to study multivalent effects across wide ranges of ligand number, density, and three-dimensional arrangement. Using L-lysine γ-substituted peptide nucleic acids, the multivalent effects of an integrin antagonist were examined over a range of 1 to 45 ligands. The optimal construct improves the inhibitory activity of the antagonist by two orders of magnitude against the binding of melanoma cells to the extracellular matrix in both in vitro and in vivo models.

Nucleic Acid-programmed Assemblies: Translating Instruction into Function in Chemical Biology

“…The recent demonstration that PNA-based assemblies are effective in vivo paves the way for their use in medical applications. [93] The use of DNA to display small molecules, peptides, glycans, and biomolecules will benefit from continued improvements in DNA sequencing and analysis technologies (forecasted to surpass Moore's progression of superconductors). This technology offers the possibility of exploring a large molecular diversity space with unprecedented speed.…”

“…Our group and others have used this strategy with a typical gain in binding affinity ranging from 10-to 1000-fold as a result of the synergic interaction of displayed ligands. [32,35,[40][41][42][43][44][45][46][47][48][49][50] Pioneering efforts in the area were reported by Kobayashi and coworkers with the oligomerization of a half slide complementary DNA fragment derivatized with galactose to generate periodic glycocluster. [51,52] Neri and coworkers were the first to use complementary sequences within the tag to pair small molecule fragments using self-hybridization.…”

Lighting Up the Force: Investigating Mechanisms of Mechanotransduction Using Fluorescent Tension Probes

“…Mechanical streptavidin-biotin dissociation was unexpected, because the reported K D (affinity) for streptavidin-biotin is at least 10 6 times greater than that of the integrin-ligand bond (119)(120)(121). In addition, dissociation rates (k off ) predict that the integrin receptors (k off ϭ 0.072 s Ϫ1 at 37°C) (122) would dissociate before streptavidin-biotin dissociation (k off ϳ 10 Ϫ5 s Ϫ1 at 37°C) (99,123,124).…”

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

“…25 Functionalizing PNA at the gamma position would also allow a peptide to be connected anywhere along the backbone or even at multiple locations to form stable cyclic PNA-peptides. 21,26 Although conjugating fluorescent dyes and other small molecules to peptides or PNA is greatly simplified when performed after synthesis while the product is still on the solid phase, the conjugation efficiency decreases for larger molecules and the cleavage and purification conditions can be detrimental to some molecules, such as metallopeptides 27 or folded peptides and proteins.…”

“…18,19 PNA has also been assembled within DNA scaffolds to investigate applications in bionanotechnology. [20][21][22] Previously we showed the versatility of using PNA linkers for assembling peptides 22 and proteins 23 into a 3D DNA nanocage. The assembly process was rapid and could be performed at or below room temperature, providing mild conditions that preserved the function of the attached polypeptides.…”