12th March 2019

Background:

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.

Figure 1. Aptamer Beacons can make use of a conjugated fluorescent dye and quencher (left) or a pair of FRET-compatible dyes (right). In either format, target binding induces a conformational change in the aptamer, changing the distance between the reporter molecules. This leads to a gain of signal or shift in fluorophore emission.

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 complimentary oligonucleotide is displaced, ‘turning on’ fluorescence in a simple, solution based, quantitative, gain of signal assay (Figure 2)

Figure 2. Aptamer Group displacement selection approach is amenable to Aptamer Beacon generation

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 the so-called 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.

Example Data:

Three different aptamer beacon assays are shown here.

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 (Figure 3). This effect is not seen when a control protein (BSA) is added.

 

Figure 3. Thrombin binding aptamer beacon shows a concentration dependent fluorescent response to its target (Thrombin, blue bars) but not the control targets (BSA, orange bars).

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 (Figure 4). This effect is concentration dependent and is not seen when other targets (ATP or GTP) are added.

Figure 4. Spinach aptamer beacon shows a concentration dependent fluorescent response to its target (ADP, blue trace) but not the control targets (ATP or GTP), orange and green respectively.

In the final example we have prepared an Aptamer Beacon using an aptamer isolated using our small molecule displacement approach. In this assay we have shown that the selected aptamer to the chemotherapeutic Imatinab, gives a clear response to the target molecule in a buffered plasma sample (Figure 5). The aptamer shows no response to buffered plasma alone. The unselected starting library shows no response to either sample.

Figure 5. A chemotherapeutic binding aptamer developed by Aptamer Group shows a clear response to its target Imatinib in plasma (right hand side, blue bars); minimal response is seen for plasma alone (right hand side, red bars). The unselected aptamer starting library control (left hand side) shows no significant response to plasma with or without the chemotherapeutic target.

Conclusion:

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. This aptamer and others will be available soon as part of our validated catalogue.

Follow up your interest in small molecule discovery, displacement assay analysis, and our new catalogue by completing the form below or contacting us at info@aptamergroup.co.uk

References:

Paige JS, Wu KY, and Jaffrey SR. RNA mimics of green fluorescent protein. Science. 2011 Jul 29;333(6042):642-6. DOI:10.1126/science.1207339

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