Oligonucleotide-Based Systems for Input-Controlled and Non-Covalently Regulated Protein-Binding

Abstract

Supramolecular chemists continuously take inspiration from complex biological systems to develop functional molecules involved in molecular recognition and self-assembly. In this regard, "smart" synthetic molecules that emulate allosteric proteins are both exciting and challenging, since many allosteric proteins can be considered as molecular switches that bind to other protein targets in a non-covalent fashion, and importantly, are capable of having their output activity controlled by prior binding to input molecules. This review discusses the foundations and passage toward the development of non-covalently operated oligonucleotide-based systems with protein-binding capacity that can be precisely regulated in an input-controlled manner.

Keywords: input-responsive; multivalency; oligonucleotide; protein binding; structure-switching; supramolecular.

Figure 1

Re-engineered N-WASP that imparts novel input-responsiveness. Intramolecular binding between a non-native PDZ domain and its peptide partner leads to a conformationally deactivated protein that is subsequently activated in the presence of excess free peptide.

Figure 2

Schematic showing the range of inter- and intra-molecular ON motifs discussed in the following section.

Figure 3

Schematic of two input responsive quadruplex-based systems designed by the Tan (A) and Mergny (B) groups, respectively. In both cases a FRET system was used to detect the structure switching. The fluorophore (F) is represented by a yellow star, and the quencher (Q) by a purple circle.

Figure 4

Schematic of two aptamer-based systems triggered upon input recognition. A “signal on” device (A) prepared by Li et al, and a “signal off” device (B) designed by Ho and colleagues resulting in increased and decreased fluorescence signals, respectively. The fluorophore (F) is represented by a yellow star, the quencher (Q) is shown as a purple circle and the input (i.e., target) as a large orange star.

Figure 5

Strategy for bivalent protein recognition based on ESAC libraries. Hybridization of two sublibraries leads to a self-assembled large library wherein each member represents a potential bidentate binder against the target protein. After biopanning, washing and removal of unbound molecules, the remaining binders are PCR-amplified, decoded and conjugated covalently to yield potential pharmacophores.

Figure 6

Representations of (A) a DNA-peptide bio-conjugate “Synbody” utilized for bivalent binding, developed by Chaput et al, as well as multivalent protein binding systems using the helical axis of DNA designed by (B) Seitz and coworkers, (C) Merkx and colleagues, and (D) Kobayashi et al.

Figure 7

Potassium cation induced self-assembly of parallel G-quadruplexes functionalized with protein-binding head-groups. (A) Iminodiacetate ligand used for the recognition and denaturation of cytochrome c. (B) Cyclic peptides harnessed to inhibit α-chymotrypsin. Note: the light blue squares represent guanine quartets.

Figure 8

Sandwhich ELISA assay featuring a bi-facial DNA pentaplex. Microwells are first coated with CRP antibody. Subsequently, CRP is exposed to the wells and then DNA-pentaplexes capable of binding both CRP and streptavidin are added. The captured CRP is detected by the addition of streptavidin-linked HRP which provides a colorimetric signal.

Figure 9

Schematic designs for a set of aptamer-based controllable protein binding agents. (A) An anticoagulant capable of deactivation through use of an “antidote”, designed by Rusconi et al (B) A toehold-exchange driven machine capable of binding and releasing thrombin via addition of two ON inputs.

Figure 10

Stimuli-responsive transcription factor binding system developed by Mascarenas et al. (A) A schematic showing the necessity of ON hybridization to cJun binding. On the right, following hybridization exposure to cJun leads to strong binding, while on the left, the ssDNA/peptide conjugate alone shows much weaker binding. (B) shows the structure of the bound system.

Figure 11

Schematic design of peptide-PNA conjugates developed by Seitz et al for protein binding controlled by (A) DNA or (B) RNA inputs.

Figure 12

A stimuli-responsive DNA chimera, designed by Jayawickramarajah et al, that can switch between monodentate and bidentate trypsin binding conformations.

Figure 13

Stimuli-responsive DNA chimera utilizing an intramolecular host-guest system on a DNA hairpin scaffold for modulating its protein binding ability.

Figure 14

Input-driven DNA-based molecular tweezer that controls the projection of a pair of thrombin binding aptamers for selective protein binding. Note: Reprinted with permission from C. Zhou, Z. Yang and D. Liu, J. Am. Chem. Soc., 2012, 134, 1416–1418. Copyright 2012 American Chemical Society.