Thermo-Programmed Synthetic DNA-Based Receptors

Abstract

Herein, we present a generalizable and versatile strategy to engineer synthetic DNA ligand-binding devices that can be programmed to load and release a specific ligand at a defined temperature. We do so by re-engineering two model DNA-based receptors: a triplex-forming bivalent DNA-based receptor that recognizes a specific DNA sequence and an ATP-binding aptamer. The temperature at which these receptors load/release their ligands can be finely modulated by controlling the entropy associated with the linker connecting the two ligand-binding domains. The availability of a set of receptors with tunable and reversible temperature dependence allows achieving complex load/release behavior such as sustained ligand release over a wide temperature range. Similar programmable thermo-responsive synthetic ligand-binding devices can be of utility in applications such as drug delivery and production of smart materials.

Keywords: DNA nanotechnology; entropy; intrinsic disorder; molecular switches; temperature-responsive nanocarriers.

Figure 1

Thermo-regulated ligand-biding receptors. Thermo-responsiveness of a synthetic bivalent receptor can be finely modulated by controlling the length (and thus entropy) of the linker connecting the two binding domains. Receptors with longer linkers (associated with higher entropy) will release their ligand at a lower temperature compared to receptors displaying the same binding domains but with shorter linkers (associated with lower entropy). This provides a way to rationally program the temperature at which a synthetic receptor loads and releases its ligand.

Figure 2

Rational design of a set of thermo-programmed triplex-forming DNA receptors. (A) (Top) Scheme of a classic bivalent molecular receptor in which two binding domains are connected by a linker domain. (Bottom) A synthetic DNA-based bivalent receptor in which two triplex-forming binding domains are connected by a poly-T linker domain. (B) Melting curves of DNA receptors sharing the same binding domains but with different lengths of the linker domain (indicated on the top of the panel). (C) 1/T vs ln(C₀) plots obtained by thermal melting curves at different equimolar concentrations (C₀) of DNA receptors and ligand. (D) Plot of ΔS vs linker length. (E) Plot of T_50% vs ΔS for the set of DNA receptors used. Melting curves experiments were performed in PBS buffer and 10 mM MgCl₂ at pH 5.5 with a temperature ramp of 1 °C·min^–1 and using a concentration of DNA receptor and ligand of 100 nM and 10 nM, respectively.

Figure 3

Thermo-programmed ligand’s load/release with triplex-forming DNA receptors. Release kinetics of triplex-forming DNA receptors with a 4-nt (A) and 60-nt (B) linker domain monitored through temperature jumps (from 25 °C to the indicated final temperatures). Three representative temperatures (50, 55, and 60 °C) were colored for better comparison. (C) Time-course experiments using two DNA receptors (4-nt in red and 60-nt in blue) in the same solution designed to bind two different 11-nt ligands (each labeled with a different fluorophore). (D, E) Load/release experiments at two temperature ranges (25 to 55 °C and 55 to 65 °C) for 4-nt (D) and 60-nt (E) triplex-forming DNA receptors. Time-course experiments (panels A and B) and load/release experiments (panels D and E) were performed in PBS buffer and 10 mM MgCl₂ pH = 5.5 with a fixed concentration of DNA receptors (4-nt and 60-nt, 100 nM each) and 11-nt ligand (10 nM). For panel E two different DNA receptors were employed with two different 11-nt ligands.

Figure 4

Thermo-programmed ATP-binding aptamer. (A) We re-engineered the ATP-binding aptamer to act as a bivalent-binding receptor by splitting its binding pocket and connecting them with a poly(T) linker domain. (B) Melting curves of ATP-binding receptors with different linker lengths. (C) Linear dependence between T_50% and ΔS values for the different receptors. (D) Time-course experiments obtained using two aptamer variants (4-nt in red and 40-nt in purple) in the same solution designed with the same binding domains for ATP but labeled with different fluorophores. (E) Load/release experiments at two temperature ranges (35 to 50 °C and 50 to 70 °C) for 4-nt and 40-nt ATP-binding aptamers. Melting curves experiments (B), time-course experiments (D), and load/release experiments (E) were performed in 100 mM Tris HCl and 10 mM MgCl₂ at pH 6.5 with a ramp of 1 °C·min^–1, with a fixed concentration of ATP-binding aptamer of 50 nM and 3 mM of ATP.

Figure 5

Extending the dynamic temperature range of ligand release. (A) Melting curves of triplex-forming DNA receptors (4-nt, 15-nt, and 60-nt linker) in the presence of ligand. (B) Extending the dynamic temperature range of ligand release by combining in the same solution a stoichiometric concentration of 4-, 15-, and 60-nt DNA receptors. (C) Time-course experiments at different temperature intervals for two receptors alone (4-nt, red and 60-nt, blue) and with three receptors (4-nt, 15-nt, and 60-nt) in the same solution (black). (D) Melting curves of ATP-binding aptamer variants (4-nt, 15-nt, and 60-nt linker) in the presence of ATP. (E) Extending the dynamic temperature range of ATP release by combining in the same solution a stoichiometric concentration of 4-, 16-, and 70-nt aptamer variants in the same solution. (F) Time-course experiments at different temperature intervals for two aptamer receptors alone (4-nt, red and 70-nt, blue) and with three variants (4-nt, 16-nt, and 70-nt) in the same solution (black). For triplex-forming DNA receptor melting curves (A), extended dynamic temperature range experiments (B), and time-course experiments (C) were performed in PBS buffer and 10 mM MgCl₂, at pH 5.5 with a fixed concentration of DNA receptors (100 nM for a single receptor and 33.3 nM each for the mixture of three receptors) and 11-nt target (10 nM). For ATP-binding aptamer receptor melting curves (D), extended dynamic temperature range experiments (E) and time-course experiments (F) were performed in 100 mM Tris HCl and 10 mM MgCl₂, at pH 6.5 with a fixed concentration of ATP-binding aptamers (50 nM for a single receptor and 16.6 nM each for the mixture of three receptors) and ATP (3 mM).