Table 4: Methods for determining aptamer binding affinity.

MethodDescription of method and applicability to small moleculesSample reference

Spectroscopy-based methods

Fluorescence intensityThe fluorescence of the aptamer or target may be quenched or increased upon binding. This method requires a fluorescent small molecule target or requires labelling of the target. [208]
Fluorescence polarizationA fluorophore is excited with polarized light and, due to rotational diffusion, the size of the fluorophore will dictate the proportion of polarized light that is emitted. This method requires a fluorescent small molecule target or target labelling. It can be used with a fluorescently tagged aptamer, however, the method is less sensitive as the overall change in mass upon binding a small molecule will be less dramatic. [209]
UV-vis absorptionThis method requires a change in intensity or wavelength of absorption in either the aptamer or target’s UV-vis spectrum. In some cases, melting studies can be used to determine . [210]
Circular dichroism (CD)CD refers to the differential absorption of left and right circularly polarized light. Upon aptamer binding to the target, the CD spectra may change but a significant difference in conformation upon target binding is required for this method to have good sensitivity. [211]
Nuclear magnetic spectroscopy (NMR)By comparing the heteronuclear single quantum coherence spectroscopy (HSQC) of individual amide protons in the free and bound aptamer, it is possible to observe changes in the chemical shifts of the peaks. This method requires conformation changes in the aptamer for good sensitivity. [212]

Mass-sensitive surface-based measurements

Surface plasmon resonance (SPR)Either the target or aptamer can be coupled to a chip; by flowing various concentrations of the nontethered ligand, changes in refractive index can be measured as the aptamer-target complex forms. If the small molecule target is immobilized, its ability to bind to the aptamer may be compromised. Immobilization of the aptamer, however, leads to a less sensitive measurement as the smaller target will cause less of a change at the surface. [33]
Quartz crystal microbalance (QCM)This method uses piezoelectric crystals to correlate the mass accumulated (target binding) on the surface with a decrease of the resonance frequency of the quartz crystal. Once again, small molecule target immobilization could affect binding affinity. Immobilization of the aptamer leads to a less sensitive measurement because there will less of a mass change upon target binding. [213]

Separation-based methods

High-performance liquid chromatography (HPLC) Zone separations of the free aptamer, target, and aptamer-target complex can be used to assess the equilibrium distribution of these components. This method is particularly difficult with small molecule targets as they have less of an effect on the separation of aptamer-target complex from the free aptamer. [214]
Capillary electrophoresis (CE), kinetic capillary electrophoresis (KCE), affinity probe capillary electrophoresis (APCE)This method is similar to HPLC except that it using an electric field to separate the components of the mixture by size and charge. Small molecule targets can be a challenge, typically requiring labeling of the small molecule although label-free KCE UV has recently been described. Once again, separation of the aptamer-target complex from the free aptamer can be more difficult in the case of small molecule targets. [64, 215, 216]
Microfree-flow electrophoresis (μ-FFE)This technique separates aptamer and aptamer-target complex based on their electrophoretic mobilities. Sample is continuously streamed into a planar flow channel while an electric field is applied perpendicularly to the direction of flow, deflecting analyte streams as they travel through the flow channel according to their mobility. Once again, this method is less effective with small molecule targets. [217]
Equilibrium dialysisEquilibrium dialysis allows the aptamer, target and the complex to equilibrate in a two compartment cell separated by a semipermeable membrane that allows only the smallest component to pass through. This method can be hampered by nonspecific adsorption of small molecule targets to the membrane.[26]
Ultrafiltration/ nitrocellulose filtrationThis method is similar to dialysis. The aptamer and target are incubated to allow binding. The fraction of the smallest unbound component is forced through a filter and measured. Once again, nonspecific adsorption to the membrane can cause this method to be unreliable. [218]
Affinity chromatographyEither the target or aptamer is covalently immobilized to a solid-phase support. The other component is incubated with the support and the amount of binding is calculated. As with other methods, chemical modification of the target or the aptamer to allow for immobilization can affect binding. [7]
Electrophoretic mobility shift assay (EMSA)The presence of the target will cause an increase in molecular weight of the aptamer-target complex, resulting in a change in electrophoretic mobility and a gel shift. This approach is not effective with small molecule targets unless a significant conformational change is observed upon binding. [219]
Optical thermophoresisBased on the directed movement of molecules along temperature gradients, the thermophoresis of an aptamer typically differs significantly from that of an aptamer-target complex because of changes in size, charge, or solvation energy. This method requires fluorescent labelling which could affect binding. Also, it could be less sensitive for small molecule aptamers due to the smaller change in mass upon target binding. [220]

Other methods

Isothermal titration calorimetry (ITC)This method allows simultaneous determination of , stoichiometry, and thermodynamic properties. It relies on the fact that formation of the aptamer-target complex is an exothermic process. Effective for small molecule aptamers, particularly if a large conformational change occurs upon target binding. [221]
High-throughput affinity quantitative PCR binding assayWith this method, an aptamer duplex is incubated with the target. The concentration of aptamer released by this binding event is then measured using real time PCR. [222]
In-line probingSpontaneous cleavage of the RNA backbone is affected by local structural characteristics, which in turn are impacted by target binding. Can be effective for small molecule aptamers but requires conformational changes upon target binding and is only applicable to RNA aptamers. [223]
Footprinting assaysThis method determines the region of aptamer sequence where target binding occurs by exploiting that the target may protect the aptamer from enzymatic cleavage/chemical reactions. Footprinting assays are easier with larger targets or require conformational changes with target binding. [224, 225]