RNA Aptamers Target NMDA Receptors

February 2015

High Affinity RNA Aptamers Target NMDA Receptors, Boasting Specificity, Flexibility and Economical Advantages

Conditions of the central nervous system are difficult to treat, particularly those targeting post-synaptic receptors. One such receptor, the N-methyl-D-aspartate (NMDA) receptor, an ionotropic glutamate receptor, is known to trigger long-lasting changes in synapses. NMDA receptor-dependent synaptic plasticity is important not only in physiological functions such as learning and memory, but also in unwanted pathological conditions such as Parkinson’s disease and chronic pain. Tight regulation of the receptors is crucial for maintaining proper function. Furthermore, subunits of the post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and kainate receptor have similar homology, making target discernment even more challenging. To address these challenges, Lee et al. have turned to aptamers.

Aptamers are nucleic acid based molecules that bind to their targets with high affinity and have typical binding dissociation constants (Kd) in the pM to nM range. They are known for their remarkable specificity and can often discern between conformations of a target molecule. Aptamers are stable under a variety of storage conditions and can recover their active tertiary structures in some conditions. In contrast to antibodies, aptamers can be synthesized quickly, economically and reproducibly. Additionally, aptamers can be modified, commonly with 2′F or 2′OMe, to increase stability and affinity.

Using Systematic Evolution of Ligands by Exponential Enrichment (SELEX), the authors started with an aptamer library of 1015 RNA sequences to screen against NMDA receptors containing the GluN1/GluN2A subunits. To increase specificity, they also went through several rounds of negative selection against GluA1 and GluK2, which are subunits of the AMPA and kainate receptors with similar homology. By the ninth round, they found several remaining aptamers contained a consensus sequence of GCGGG. From this pool they identified one clone, C26, to have a significantly higher affinity with a Kd of 65 ± 3 nM.

The authors then carried out a number of experiments examining the affinity and specificity of C26 and two truncated forms, termed C26-31 and C26-50. While C26-31 demonstrated substantially decreased affinity to GluN1/GluN2 and was not specific, both C26 and C26-50 continued to perform well. Interestingly, C26-50 showed a persistent blockage of NMDA receptor currents in the presence of saturating amounts of glutamate, suggesting that it’s a non-competitive inhibitor. Further testing confirmed this hypothesis.

Finally, the authors tested whether these aptamers contained neuroprotective properties. Using an oxygen-glucose deprivation model of neuronal death, they found that C26-50 maintained cell viability at concentrations as low as 250 nM. This result is similar to the FDA-approved small molecule drug, memantine. The authors note, however, that the development of aptamers is often quicker and cheaper than small molecules, owed in large part to the fact that no structural information is needed.

TriLink supplied the 2′F or 2′OMe NTPs used in this research. In addition to being the original manufacturers of many modified nucleotides used to impart nuclease resistance, 

Have a question? Visit Ask An Expert.