Aptamer Beacons

‘Aptamer beacons’ are an alternative, fluorescence-based assay format, used to detect interaction between an aptamer and its target.

This assay format has a number of advantages:

  • Simple solution based assay format – add sample, mix, read – no complicated incubations, wash steps etc. are required
  • Field-based application using a simple hand-held reader
  • Multiplexed Assays – conjugation of aptamers with a variety of fluorophores simplifies development of multiplexed assays

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.

In an Aptamer Beacon, the aptamer will be labelled with a fluorophore and a quencher (to give a gain-of-signal) or a pair of FRET-compatible 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; 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 (i.e. a change in fluorescence colour).

A wide variety of fluorophores are available enabling simple development of multiplexed assays.

Aptamer Beacons allow simple, solution-based detection of analytes

Aptamer Beacons 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, altering the distance between the reporter molecules. This leads to a gain-of-signal or shift in fluorophore emission.

Aptamer Beacon Formats

Aptamer Beacons can be developed in a wide variety of formats. Many of these are reviewed in Aptamer binding assays for proteins: the thrombin example – a review.

At Aptamer Group, our displacement-based small molecule aptamer selection approach is especially amenable to the development of Aptamer Beacons of the type shown in example ‘D’.

A complimentary oligonucleotide labelled with a quencher (or FRET pair) can be hybridised to our aptamers ‘turning off’ fluorescence. Upon binding to the small molecule target, the conformational change in the aptamer leads to ‘elution’ of the complimentary oligonucleotide, ‘turning on’ fluorescence in a simple, solution-based, quantitative, gain-of-signal assay.

Small molecule binding aptamers developed using Aptamer Group displacement approach are readily used in an Aptamer Beacon format

A chemotherapeutic binding aptamer shows a clear response to its target in a plasma (green bars); minimal response is seen for plasma alone (red bars). The unselected aptamer starting library control shows no significant response to plasma with or without the chemotherapeutic target.

Aptamer Beacons allow simple, solution-based, concentration dependent detection of analytes

A thrombin-responsive Aptamer Beacon was created by attaching a fluorophore and quencher to either end of the thrombin aptamer. Thrombin binding induces a conformational change in the Aptamer Beacon, restoring fluorescence in a concentration dependent manner (green bars). No response is seen for the BSA control protein (red bars).

Spinach-Based Aptamer Beacons

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-based aptamer beacons’.

In Spinach-based Aptamer Beacons, the aptamer binds the fluorophore (DFHBI) and stabilises the fluorescent conformation of the dye (a). The reporting ‘Spinach module’ is combined with a sensing ‘Recognition module’ such that the Spinach module structure is disrupted until the target binds to the Recognition module (b). This binding event leads to a conformational change which restores the fluorophore binding aptamer structure and hence restores fluorescence (c).
An example Spinach aptamer beacon was created using an ADP binding aptamer as the ‘Recognition module’. Binding of ADP to the construct, restores the active fold of the ‘Spinach module’ and allows binding of the fluorophore and the associated increase in fluorescence. This effect is ADP concentration dependent (blue) and is not seen when other targets (ATP or GTP) are added (orange and green respectively).

References:

  1. Anal Chim Acta. 2014 Jul 21;837:1-15. doi: 10.1016/j.aca.2014.04.055. Epub 2014 May 2. Aptamer binding assays for proteins: the thrombin example–a review. Deng B1, Lin Y2, Wang C1, Li F1, Wang Z1, Zhang H1, Li XF1, Le XC3.
  2. 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