Nanopore Detection of Single‐Molecule Binding within a Metallosupramolecular Cage

Abstract

Guest encapsulation is a fundamental property of coordination cages. However, there is a paucity of methods capable of quantifying the dynamics of guest binding processes. Here, we demonstrate nanopore detection of single-molecule binding within metallosupramolecular cages. Real-time monitoring of the ion current flowing through a transmembrane α-hemolysin nanopore resolved the binding of different guests to both cage enantiomers. This enabled the single-molecule kinetics of guest binding to be quantified, whereas the ordering and durations of events were consistent with a guest-exchange mechanism that does not involve ligand dissociation. In addition to providing a new approach for single-molecule interrogation of dynamic supramolecular processes, this work also establishes that cage complexes which are too large to enter the nanopore can be exploited for detecting small molecules, thus constituting a new class of molecular adapter.

Teaching indicators to unravel the kinetic features of host–guest inclusion complexes

“…10,11 The design of out-of-equilibrium systems also requires knowledge of both K a values and rate constants. [12][13][14][15] However, except for CEST-active 3 or slowly equilibrating systems that can be monitored by NMR (e.g., DOSY, EXSY, inversion recovery), 1,[16][17][18][19][20] kinetic rate constants of supramolecular systems are experimentally mostly only available for chromophoric or emissive systems. 2,4,[21][22][23] These experiments are typically conducted as time-resolved direct host-guest binding titration assays, herein abbreviated as kinDBA (Fig.…”

“…In some cases, single molecule measurements with nanopores allowed for assessing the kinetic rate constants for complexation and decomplexation of entrapped host-guest complexes. 15,24,25 Conversely, binding affinities (K a ) of host-guest complexes can be obtained for a wide range of hosts and guests by several different techniques, for instance, through NMR titrations and calorimetric measurements (ITC) as representative directbinding assays [26][27][28] or competitive-binding assays such as the indicator-displacement assay (IDA) 28,29 and the recently by us introduced guest-displacement assay (GDA). 30 Consequently, there is a strong mismatch between the number of reported binding affinities and kinetic parameters for any class of hostguest complexes.…”

Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms

“…The popularity of this nanopore is likely due to its robustness and well-established structure. Although modification of α-HL via mutagenesis is possible, ,,,, the commercial availability of α-HL means that it is often used in its wild-type form. ,,− ,,, Furthermore, cysteine mutants may sometimes present purification or misfolding issues arising from disulfide formation. Recently, fully assembled wild-type α-HL nanopores have been modified in situ through direct chemical functionalization of lysine residues .…”

“…Analytical techniques based on transmembrane protein nanopores have become well-established methods for investigating chemical and physical phenomena on the single-molecule level. − Although less physically robust than solid-state nanopores, , biological nanopores offer the advantage of having atomically precise structures, thereby providing superior precision and reproducibility in their applications. ,, Monitoring the current flow through a nanopore under an applied transmembrane potential (voltage) enables the real-time, in situ characterization of the interaction between the pore and other molecular entities: changes in the recorded current can provide information about the molecular structure of an analyte, − the kinetics and dynamics of noncovalent binding, ,− or chemical reactivity. − Biological nanopores have even served as the scaffold for constructing molecular machines. − …”

Orthogonal binding and displacement of different guest types using a coordination cage host with cavity-based and surface-based binding sites

“…Coordination cages – hollow, pseudo-spherical metal/ligand assemblies – are well known to be able to bind small molecular guests in their central cavities, 1 with a range of consequences for, and potential applications in, areas such as catalysis, 2 sensing 3 and transport. 4 The focus on guest binding has mostly been occupancy of these central cavities, as they present an obvious binding site with well-defined size, shape and (sometimes) functional group characteristics which obviously relate to their molecular recognition properties.…”