Aptamers as positive modulators

What to do when you want to validate an assay for drug discovery, but there are little or no literature tools available?

Well according to a publication from a group at Astra Zeneca, the use of Aptamers could be one way to solve this problem. Aptamers are lengths of DNA or RNA, generally 20-100 bases, which could be used in the same manner as small molecule tools, binding to targets of interest.

Using the historically difficult target glycine receptor as a model system (GlyRα1), the authors  generated aptamer libraries of RNA using the SELEX methodology (systematic evolution of ligands via exponential enrichment). This process involves the exposure of an aptamer library to your target, which has been immobilised (here the team used a biotin/streptavidin interaction), any unbound material is washed away and bound material is collected and amplified by RT-PCR. This is repeated across a number of iterations, improving the overall success rate.

In this case the SELEX process was run with a variety of different sources of the glycine receptor.  The active molecules generated from this method were then further validated using a radioligand filter binding assay. This resulted in eight aptamers being selected for scale up and further profiling in a variety of glycine receptor assays.

Selective binding of the aptamers was shown using SPR using immobilized glycine receptor, this was further supported by immunofluorescence of fixed cells and live cell imaging experiments.  Functional profiling of the aptamers occurred using a membrane potential dye assay supported with patch clamp electrophysiology.

The SPR measurements revealed all eight aptamers had Kd values in the low nanomolar range.  Interestingly two of the eight aptameters had slow on and off rates of binding .The cellular locations from the imaging experiments showed that the majority of the aptamer was present in the cytosol, and to a lesser extent at the plasma membrane. The authors suggested the cytosol accumulation may be due to interactions with Golgi and endoplasmic reticulum.

The functional assays highlighted some interesting findings. Using the membrane potential dye assay, five of the aptamers gave results suggesting they were positive modulators of the glycine receptor.  When this was further explored with one of the aptamers (c2) in a single cell patch clamp it was shown to be a positive modulator.

Overall the publication uses a variety of different supporting techniques to identify a positive modulator aptamer of the glycine receptor.

Could these molecules have a brighter future than just tools and become an alternative to small molecule therapeutics?  The issue of stability and delivery of the treatment have to be solved in each case, but the answer is yes.  As the authors point out, the FDA has approved the aptamer Macugen used for the treatment of age related macular degeneration.  The significant drawback of this medication is the requirement that it has to be injected into the eye of the patient.  So for the moment, tools seem to the current use for aptamers, however other clinical uses will be developed.


Figure showing the positive modulation of glycine receptor using aptamer C2

Shalaly, N. D., Aneiros, E., Blank, M., Mueller, J., Nyman, E., Blind, M., Dabrowski, M. A., et al. (2015). Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers. Journal of Biomolecular Screening, 20(9), 1112-1123. Retrieved from http://jbx.sagepub.com/cgi/doi/10.1177/1087057115590575


Blog written by Gareth Williams


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