Aptamer Modification

Aptamers can be directly conjugated to reporter molecules at any point of the sequence, allowing easy incorporation into your assay of choice

One of the most useful (and readily available) modifications is the incorporation of a wide variety of fluorophores. These can be added to either the 5′ or 3′ ends of the aptamer, or at any given point along its length. This makes it a trivial matter to monitor aptamer-target interactions by monitoring changes in the aptamer fluorescence properties such as fluorescence polarisation or anisotropy.

Fluorescent aptamers also allow direct visualisation of the target, so they may be used as a more specific alternative to antibodies in techniques such as Western blotting. Aptamers would have many advantages in this sort of application, the most obvious of which is speed. As aptamers are highly specific for their target, they may be used directly after transfer to a membrane. They would negate the need for ‘membrane blocking’, secondary antibodies or additional chemiluminescent reagents. As many different fluorophores are available, multiple targets could be identified in a single sample.

Aptamers may also be directly visualised in tissue samples or be used to follow binding and cellular localisation by confocal microscopy. The wide range of available fluorophores also means that multiple aptamers can be used in a single sample allowing co-localisation studies. Labelled aptamers may also be used for FACS analysis allowing identification of specific cell types or cellular markers within mixed cell populations.

An extension of this is the conjugation of ‘FRET pairs’ or ‘fluorophore-quencher’ pairs to the selected aptamer. Target binding results in a significant conformational change in the aptamer, which in turn changes the distance between the FRET pairs (or fluorophore and quencher) resulting in a significant change in fluorescent output. This produces so called ‘aptamer beacons’.