Functionalised nanopores: chemical and biological modifications.

Nanopore technology has established itself as a powerful tool for single-molecule studies. By analysing changes in the ion current flowing through a single transmembrane channel, a wealth of molecular information can be elucidated. Early studies utilised nanopore technology for sensing applications, and subsequent developments have diversified its remit. Nanopores can be synthetic, solid-state, or biological in origin, but recent work has seen these boundaries blurred as hybrid functionalised pores emerge. The modification of existing pores and the construction of novel synthetic pores has been an enticing goal for creating systems with tailored properties and functionality. Here, we explore chemically functionalised biological pores and the bio-inspired functionalisation of solid-state pores, highlighting how the convergence of these domains provides enhanced functionality.

Switchable foldamer ion channels with antibacterial activity.

Synthetic ion channels may have applications in treating channelopathies and as new classes of antibiotics, particularly if ion flow through the channels can be controlled. Here we describe triazole-capped octameric α-aminoisobutyric acid (Aib) foldamers that "switch on" ion channel activity in phospholipid bilayers upon copper(ii) chloride addition; activity is "switched off" upon copper(ii) extraction. X-ray crystallography showed that CuCl2 complexation gave chloro-bridged foldamer dimers, with hydrogen bonds between dimers producing channels within the crystal structure. These interactions suggest a pathway for foldamer self-assembly into membrane ion channels. The copper(ii)-foldamer complexes showed antibacterial activity against B. megaterium strain DSM319 that was similar to the peptaibol antibiotic alamethicin, but with 90% lower hemolytic activity.

Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms.

Nanopores are emerging as a powerful tool for the investigation of nanoscale processes at the single-molecule level. Here, we demonstrate the methionine-selective synthetic diversification of α-hemolysin (α-HL) protein nanopores and their exploitation as a platform for investigating reaction mechanisms. A wide range of functionalities, including azides, alkynes, nucleotides, and single-stranded DNA, were incorporated into individual pores in a divergent fashion. The ion currents flowing through the modified pores were used to observe the trajectory of a range of azide-alkyne click reactions and revealed several short-lived intermediates in Cu(I)-catalyzed azide-alkyne [3 + 2] cycloadditions (CuAAC) at the single-molecule level. Analysis of ion-current fluctuations enabled the populations of species involved in rapidly exchanging equilibria to be determined, facilitating the resolution of several transient intermediates in the CuAAC reaction mechanism. The versatile pore-modification chemistry offers a useful approach for enabling future physical organic investigations of reaction mechanisms at the single-molecule level.

Enzymatically Ligated DNA-Surfactants: Unmasking Hydrophobically Modified DNA for Intracellular Gene Regulation.

Herein, we describe the characterization of a novel self-assembling and intracellular disassembling nanomaterial for nucleic acid delivery and targeted gene knockdown. By using a recently developed nucleic acid nanocapsule (NAN) formed from surfactants and conjugated DNAzyme (DNz) ligands, it is shown that DNz-NAN can enable cellular uptake of the DNAzyme and result in 60 % knockdown of a target gene without the use of transfection agents. The DNAzyme also exhibits activity without chemical modification, which we attribute to the underlying nanocapsule design and release of hydrophobically modified nucleic acids as a result of enzymatically triggered disassembly of the NAN. Fluorescence-based experiments indicate that the surfactant-conjugated DNAzymes are better able to access a fluorescent mRNA target within a mock lipid bilayer system than the free DNAzyme, highlighting the advantage of the hydrophobic surfactant modification to the nucleic acid ligands. In vitro characterization of DNz-NAN's substrate-cleavage kinetics, stability in biological serum, and persistence of knockdown against a proinflammatory transcription factor, GATA-3, are presented.

Nucleic Acid Nanocapsules for Enzyme-Triggered Drug Release.

Herein we describe a nucleic acid functionalized nanocapsule in which nucleic acid ligands are assembled and disassembled in the presence of enzymes. The particles are fully degradable in response to esterases due to an embedded ester cross-linker in the particle's core. During synthesis the nanocapsules can be loaded with hydrophobic small molecules and post self-assembly undergo covalent cross-linking using copper catalyzed click chemistry. They can then be functionalized with thiolated DNA through stepwise thiolyne chemistry using UV light irradiation. Additionally, the capsule is compatible with enzyme mediated functionalization of a therapeutic mRNA-cleaving DNAzyme at the particle's surface. The resulting particle is highly stable, monodisperse in size, and maximizes the therapeutic potential of both the particles interior and exterior.