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 studying 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 1-45 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.

Figure 1

Strategies to construct multivalent assemblies. a. Step-by-Step syntheses afford an accurate valency but are limited to low numbers of ligands. b. Shotgun methods provide high valencies but an error (ε) is always associated with the number of ligands. c. Scaffolds made using L-lysine γ-substituted peptide nucleic acid (^LKγ-PNA) and nucleic acid (NA) allow access to multivalent assemblies with both high valencies and accurate ligand number. Valency of an ^LKγ-PNA-NA assembly is the product of the number of complementary binding sites incorporated into the NA (x) and the number of ligands displayed from each PNA oligomer (y).

Figure 2

Chemical and cartoon representations of ^LKγ-PNA assembled onto DNA. a. Chemical structure of ^LKγ-PNA bound to DNA. b. and c. Ribbon and cartoon diagrams of four ^LKγ-PNAs (each bearing one ligand) bound to a linear DNA.

Figure 3

Construction and screening of the DNA:PNA-Y_x multivalent library. a. Generation of the multivalent library by complexing each PNA in the first box with every DNA in the second box. PNA-A is an aegPNA oligomer with a c(RGDfK) bound to the N terminus. PNA-B, PNA-C and PNA-D have 1, 2 or 3 c(RGDfK) units respectively, conjugated via γ-sidechains of internal ^LKγ-PNA residues. DNA is numbered according to how many complementary sequences each contains and represents the number of PNAs that would be complexed. Inhibition of C32 cell adhesion to vitronectin was examined with DNA:PNA complexes. b. Three-dimensional representations of IC₅₀ data from the screen of the library (A/B represents results from either PNA A or B). Data for DNA:PNA-C/D_13–15 were not acquired because inhibitory activity was maximized at shorter lengths of ssDNA (see Supplementary Fig. S2 for error bars).

Figure 4

Atomic-scale computational model of an ^LKγ-PNA:DNA complex bound toα_Vβ₃ integrin receptors. a. and b. The side and top views of a molecular model of DNA:PNA-D₁ bound to a cluster of three integrins (colored in pewter). The DNA template is dark blue, the ^LKγ-PNA (including all bases and linkers are sky blue), and c(RGDfK) residues are fuchsia.

Figure 5

in vivo activity and determination of stoichiometry for ^LKγ-PNA:DNA complexes. a. Displacement of ¹²⁵I-Echistatin from integrin α_Vβ₃ on C32 cells by c(RGDfK) and DNA:PNA-D₅. K_d c(RGDfK) = 6.3 × 10⁻⁸ M; K_d DNA:PNA-D₅= 1.6 × 1⁻¹⁰ M. Error bars represent two s.d. (n=3). b. The effect of DNA:PNA-D₅ on metastatic potential of B16F10 cells based on the tumor development in C57BL/6NCrmice. All mice were injected with 5×10⁵ B16F10 cells. Error bars represent one s.d. (n=8 mice). c. Lungs of sacrificed mice after fixation with tumor lesions indicated by dark spots. The mice treated with DNA:PNA-D₅ had visibly fewer tumor colonies present after 14 days compared to the control or c(RGDfK) alone. Each row corresponds to three mice out of a group of eight. d. Absorbance c(s) distributions obtained from sedimentation velocity data collected at 50 krpm and 20.0 °C for DNA:PNA-B₅ at loading concentrations of 0.35 (blue), 0.78 (red) and 1.47 (green) A₂₆₀. The complex was prepared using a slight excess of PNA seen at ~1.0 S.

Figure 5

in vivo activity and determination of stoichiometry for ^LKγ-PNA:DNA complexes. a. Displacement of ¹²⁵I-Echistatin from integrin α_Vβ₃ on C32 cells by c(RGDfK) and DNA:PNA-D₅. K_d c(RGDfK) = 6.3 × 10⁻⁸ M; K_d DNA:PNA-D₅= 1.6 × 1⁻¹⁰ M. Error bars represent two s.d. (n=3). b. The effect of DNA:PNA-D₅ on metastatic potential of B16F10 cells based on the tumor development in C57BL/6NCrmice. All mice were injected with 5×10⁵ B16F10 cells. Error bars represent one s.d. (n=8 mice). c. Lungs of sacrificed mice after fixation with tumor lesions indicated by dark spots. The mice treated with DNA:PNA-D₅ had visibly fewer tumor colonies present after 14 days compared to the control or c(RGDfK) alone. Each row corresponds to three mice out of a group of eight. d. Absorbance c(s) distributions obtained from sedimentation velocity data collected at 50 krpm and 20.0 °C for DNA:PNA-B₅ at loading concentrations of 0.35 (blue), 0.78 (red) and 1.47 (green) A₂₆₀. The complex was prepared using a slight excess of PNA seen at ~1.0 S.