Team:INSA-Lyon/Aptamers
Introducing aptamers
Aptamers are small single stranded nucleic acids. They have been developed since the late 80s, but their importance has started growing in the recent years. More and more small-size companies are being created and specialize in selling aptamer-based solutions. They are mainly used in two domains: in therapeutics, where they can act as enzymes inhibitors; and in detection, where they are mainly used as an alternative to antibodies. Their sequences are obtained through a method called SELEX, which is basically directed sequence evolution. Thus obtaining an aptamer against a new target is far easier than getting an antibody against the same target.
Thanks to their obtention method, aptamers are very sensitive to a particular target. Targets can be big molecules such as proteins, but also more surprisingly against small targets (ATP for instance). A new trend appeared in the recent years, with aptamers being used as detection devices against whole cells. For instance, cancerous cells could be selectively targeted. Sensitivities varies from 100 µM to a nM range. Selectivity is a more difficult question because it is always hard to guarantee that aptamers will be aimed at a single target. Tests could be made with similar structure targets to assess selectivity, but only “real life” tests can demonstrate a sufficient selectivity.
In our project
We’ve chosen to work on several targets, with two purposes, making a proof of concept and detecting STIs targets.
Adenosine aptamer (eg ATP, AMP)
Thrombin aptamers: Thrombin is the uttermost studied target with aptamers. So we wanted to use it as a proof of concept to test our techniques before starting to develop STIs aptamers. There are actually three well-described aptamers against thrombin.
HIV-1 Reverse Transcriptase (RT) aptamer is aimed at detecting circulating HIV-1 RT
HBsAg aptamer, targeted a surface antigen of the hepatitis B virus.
Characterization and reproducibility
There is a wide range of methods used to quantify affinity and selectivity of aptamers against a target. Sadly enough, for a mere iGEM team most of them are extremely costly and complicated to perform. So we’ve chosen two simple methods, that have their limitations.
Detection through fluorescence reporting. A fluorophore is fixed on the aptamer, this fluorophore is quenched if nothing happens, but if the target is present it goes away and a fluorescent signal is measurable. This method has the advantage of being fast and easy. But structural alteration of the aptamers may alter their sensitivity.
Electrophoretic Mobility Shift Assays. Basically a complex between an aptamer and its target will migrate less on a PAGE than free DNA. This property is used to quantify the interaction strength. But the gels environment may be disruptive and lower the apparent sensitivity. Anyway this technique is one of the mostly used thanks to its cost effectiveness.
