Multivalent Ultrasensitive Interfacing of Supramolecular 1D Nanoplatforms

Abstract

Multivalent display on linear platforms is used by many biomolecular systems to effectively interact with their corresponding binding partners in a dose-responsive and ultrasensitive manner appropriate to the biological system at hand. Synthetic supramolecular multivalent displays offer a matching approach for the modular and bottom-up construction and systematic study of dynamic 1D materials. Fundamental studies into multivalent interactions between such linear, 1D materials have been lacking because of the absence of appropriate modular nanoplatforms. In this work we interfaced two synthetic multivalent linear nanoplatforms based on a dynamic supramolecular polymer, formed by hybrid discotic-oligonucleotide monomers, and a series of complementary DNA-duplex-based multivalent ligands, also with appended short oligonucleotides. The combination of these two multivalent nanoplatforms provides for the first time entry to study multivalent effects in dynamic 1D systems, of relevance for the conceptual understanding of multivalency in biology and for the generation of novel multivalent biomaterials. Together the two nanoscaffolds provide easy access to libraries of multivalent ligands with tunable affinities. The DNA scaffold allows for exact control over valency and spatial ligand distribution, and the discotic supramolecular polymer allows for dynamic adaptation and control over receptor density. The interaction between the two nanoplatforms was studied as a function of ligand interaction strength, valency, and density. Usage of the enhancement parameter β allowed quantification of the effects of ligand valency and affinity. The results reveal a generalized principle of additive binding increments. Receptor density is shown to be crucially and nonlinearly correlated to complex formation, leading to ultrasensitive responses. The results reveal that, not unlike biomolecular signaling, high density multivalent display of receptors is crucial for functionally increased affinities.

Figure 1

(a) Chemical structures of (i) DNA-overhang-functionalized receptor monomers (4-Disc and 5-Disc), where the DNA overhangs are shown as light gray curved lines on the green discotic scaffold, and (ii) unfunctionalized monomer (Inert-Disc). In water, the discotic monomers self-assemble into columnar stacks, referred to here as receptor nanoscaffold. (b) Schematic representation of the receptor–ligand complex formed by the receptor nanoscaffold (made out of the discotics) and the DNA-duplex-based ligands. The ligand (mL_n) and its corresponding components are shown: in yellow the branches (mx and my) and in dark gray the backbones (B_n). “n” denotes the valency of the ligands, while “m” represents the number of complementary A-T base pairs between the DNA overhangs. The backbone units are made of sequences x′ and y′ which are complementary to the branches mx and my, respectively. The complementary base pairs between ligand/receptor and backbone/branches are drawn with black dashed lines. (c) Specific DNA sequences used for the DNA-functionalized discotic monomers (4-Disc and 5-Disc) and exemplary DNA-based ligands of series I (m = 4) and series II (m = 5). The DNA sequences of branches 4x and 5x are shown in Figure S2.

Figure 2

(a) Schematic representation of the Cy3-functionalized discotic receptor building block and nanoscaffold, and the BHQ-2-functionalized ligand nanoscaffold (series I and II respectively feature four and five bases of complementarity). Binding of the ligand to the receptor via duplex formation between the overhangs results in quenching of the Cy3-dye fluorescence. (b) Titration curves of series I ligands (4L_2,4L₃, 4L₄, 4L₅, 4L₆) to the receptor nanoscaffold formed by the 4-Disc (10 nM). (c) Titration curves of series II ligands (5L₂, 5L₃, 5L₄, 5L₅, 5L₆) to the receptor nanoscaffold formed by the 5-Disc (10 nM). The assay concentration conditions limit the evaluation of binding affinities below 1 nM (see 5L₆, yellow line). (d) Gibbs free energies (ΔG°) for each ligand–receptor complex formation plotted against the valency of the ligand (L_n) and linear fit for each series.

Figure 3

(a) Schematic representation of receptor nanoscaffold with different densities of DNA overhangs. Note that the total concentration of receptor buildings blocks with DNA overhangs (i.e., 5-Disc) was kept constant and that the density was controlled by the addition of Inert-Disc. (b–f) Titration curves of ligands 5L₂–5L₆ from series II with five complementary base pairs to the receptor nanoscaffolds with differing receptor density (for color coding see (a)). The concentration of 5-Disc was constant at 10 nM. β enhancement factors calculated with eq 1 (see also Table S3), plotted against (g) the ligand valency (n) and (h) receptor density (Θ_R).