Aptamer Beacons As A Simple Solution-Based Diagnostic Platform
Molecular Aptamer Beacons are novel reagents capable of simple, solution-based detection and reporting of target concentration through a fluorescence-based readout.
This assay format has a number of advantages
- Simple solution-based assay format – no complicated incubations, wash steps etc. are required
- Field-based application using a simple hand-held reader
- Readily developed into multiplexed assays using aptamers with a variety of fluorophores
Aptamer Beacons rely on the conformational change that occurs when most aptamers bind to their targets. Assays of this nature are extremely difficult to develop using more ‘rigid’ affinity ligands such as antibodies, peptides, Molecular Imprinted Polymers (MIPS) etc.
Generally, the aptamer will be labelled with a fluorophore and a quencher (to give a gain of signal) or a FRET pair of fluorophores (to give a colour change). Upon target binding, the aptamer undergoes a conformational change; altering the distance between the two labels. In the case of a fluorophore and a quencher, the conformational change reduces the ability of the quencher, to act upon the fluorophore. This restores the fluorescent signal, leading to a gain-of-signal. In the case of a FRET pair, the conformational change in the aptamer changes the distance between the two fluorophores and thereby alters the energy transfer between them. This results in a shift in fluorescence emission from one fluorophore to the other and a change in fluorescence colour.
At Aptamer Group, our displacement-based small molecule aptamer selection approach is especially amenable to the development of Aptamer Beacons. This approach relies on a target induced conformational change to release the aptamer from the support. A complimentary oligonucleotide labelled with a quencher (or FRET pair) can be hybridised to our fluorescently labelled aptamers ‘turning off’ fluorescence. Upon binding to the small molecule target, the complementary oligonucleotide is displaced, ‘turning on’ fluorescence in a simple, solution-based, quantitative, gain-of-signal assay.
An alternative approach to creating Aptamer Beacons is to use a target induced conformational change in one aptamer, to affect the fold and hence function, of an adjoining fluorophore-binding aptamer. These tandem aptamer systems are most commonly used to detect and quantify small molecules. One of the best-known examples of this approach are Spinach aptamer beacons.1 In this system, the Spinach aptamer binds the fluorophore DHBI and stabilises the fluorescent conformation of the dye. This ‘reporting aptamer domain’ was combined with a ‘sensing aptamer domain’ in such a way, that the Spinach aptamer structure was disrupted until the target binds to the sensing domain. This binding event leads to a conformational change which restores the fluorophore binding aptamer structure and hence restores fluorescence.
Aptamer Beacons in Action
Three different styles of aptamer beacon assays are shown here.
- A fluorescent single-reagent assay for thrombin
- Spinach aptamer beacons
- Small molecule aptamer beacon
Single-reagent thrombin assay
In the first, a fluorophore and quencher were attached to the ends of a thrombin binding aptamer. Interaction between the aptamer and thrombin results in a conformational change in the aptamer, which changes the distance between the fluorophore and quencher; restoring fluorescence. This effect is concentration-dependent as more target binds to more aptamer and therefore leads to a greater fluorescence output. This effect is not seen when a control protein (BSA) is added.
Spinach aptamer beacon
In the Spinach aptamer beacon, the Spinach aptamer is combined with an ADP binding aptamer. Binding of the construct to ADP restores the active fold in the Spinach aptamer domain and allows binding of the fluorophore and the associated increase in fluorescence. This effect is concentration-dependent and is not seen when other targets (ATP or GTP) are added.
Small molecule aptamer beacon
An Aptamer Beacon was developed using an aptamer isolated using our small molecule displacement approach. Results showed that the selected aptamer to the chemotherapeutic Imatinib gives a clear response to the target molecule in a buffered plasma sample, while the aptamer shows no interaction with the complex plasma matrix, indicating the specificity of the aptamer for the target.
These assays show the versatility of aptamer beacons and specifically demonstrates that Aptamer Group displacement selection approach creates aptamers that can be used as Aptamer Beacons in a simple fluorescence-based gain of signal assay.
Follow up your interest in small molecule discovery, displacement assay analysis, and our new catalogue by getting in touch.