A simple aptamer based assay involves the use of ‘aptamer beacons’. This is an individual aptamer sequence carrying a fluorescent dye at one end and a quencher or FRET pair ‘acceptor’ dye at the other. Binding of the aptamer to the target results in a conformational change in the aptamer which changes the distance between the ends and therefore a change in fluorescence output. This may also be achieved using a dye labelled aptamer and quencher labelled complimentary oligonucleotide, where target binding results in displacement of the complimentary strand.
For example, affinity binding of the aptamer to thrombin, and the aptamer’s structure switching upon binding, was used for developing molecular beacon approach (Figure 1).
The typical function of aptamer beacons:
- A single-stranded DNA sequence containing complementary bases at the 5′ and 3′ ends and each end is labelled with either a fluorophore or a quencher.
- A stem-loop structure is formed within the molecule and the resulting close proximity of the two ends lead to fluorescence quenching.
- When a target with complementary sequences to the loop is present, hybridization between the target sequence and the loop separates the fluorophore and the quencher, altering the fluorescence resonance energy transfer (FRET) and generating fluorescence to be detected.
This process often requires post selection modification and optimization of target specific aptamers to achieve the beacon effects. We are developing proprietary processes to directly select functional aptamer beacons to bypass the need for aptamer optimization to give a functional aptamer beacon.
An aptamer beacon assay was constructed for thrombin with BSA used as a negative control.
As illustrated in Figure 2, there is a gradual increase in fluorescence output with increased thrombin concentration. No such fluorescence is observed for the BSA negative control.
Importantly, there is no significant difference in fluorescent output between 5 seconds and 5 minutes shaking. This demonstrates the ability of the aptamer to bind to the target within a matter of seconds and give a complete read out; an important factor in many healthcare diagnostic applications.