Identification and characterization of RNA aptamers: A long aptamer blocks the AMPA receptor and a short aptamer blocks both AMPA and kainate receptors

Abstract

AMPA and kainate receptors, along with NMDA receptors, represent different subtypes of glutamate ion channels. AMPA and kainate receptors share a high degree of sequence and structural similarities, and excessive activity of these receptors has been implicated in neurological diseases such as epilepsy. Therefore, blocking detrimental activity of both receptor types could be therapeutically beneficial. Here, we report the use of an in vitro evolution approach involving systematic evolution of ligands by exponential enrichment with a single AMPA receptor target (i.e. GluA1/2R) to isolate RNA aptamers that can potentially inhibit both AMPA and kainate receptors. A full-length or 101-nucleotide (nt) aptamer selectively inhibited GluA1/2R with a K I of ϳ5 M, along with GluA1 and GluA2 AMPA receptor subunits. Of note, its shorter version (55 nt) inhibited both AMPA and kainate receptors. In particular, this shorter aptamer blocked equally potently the activity of both the GluK1 and GluK2 kainate receptors. Using homologous binding and whole-cell recording assays, we found that an RNA aptamer most likely binds to the receptor's regulatory site and inhibits it noncompetitively. Our results suggest the potential of using a single receptor target to develop RNA aptamers with dual activity for effectively blocking both AMPA and kainate receptors.Ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the mammalian central nervous system (CNS) and are indispensable for brain development and function (1, 2). The ionotropic glutamate receptor family includes three receptor subtypes that can all be activated upon binding of glutamate: ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptors (1-3). Compared with NMDA receptors, AMPA and kainate receptors are more alike in both sequence and structure (3, 4). AMPA receptors are expressed post-synaptically and are involved in fast excitatory neurotransmission (1). Kainate receptors are expressed both pre-and post-synaptically, and contribute to excitatory neurotransmission and modulate network excitability by regulating neurotransmitter release (5, 6).AMPA and kainate receptors are widely expressed in the brain. AMPA receptors have four subunits, i.e. GluA1-4. GluA1-3 are enriched in the hippocampus, outer layers of the cortex, olfactory regions, lateral septum, basal ganglia, and amygdala, etc. (7,8). The expression of the GluA4 subunit is low to moderate throughout the CNS, except in the reticular thalamic nuclei and the cerebellum where its level is high (9 -11). Kainate receptors have five subunits, i.e. GluK1-5. At the mRNA level, GluK1 is highly abundant in the neocortex, hypothalamus, and the hindbrain, whereas GluK2 is highly abundant in the cerebellum. GluK3 is widely distributed in the brain. GluK4 is enriched in the hippocampus (CA3 pyramidal cells).GluK5 is abundant in the neocortex, hippocampus (dentate gyrus and CA2, 3 pyramidal cells), and cerebellum (granule cells) (12,13). At the pro...

Aptamers: Uptake mechanisms and intracellular applications

“…One group found a full-length aptamer that blocked AMPA receptors specifically, but a truncated version of the same aptamer equally inhibited both the AMPA and kainite receptors. [125,126], suggesting that the aptamer could serve multiple functions after truncation. Even though the dual function of this aptamer might be clinically beneficial, the functional or structural changes of aptamers after truncation should be carefully investigated.…”

One aptamer, two functions: the full-length aptamer inhibits AMPA receptors, while the short one inhibits both AMPA and kainate receptors

“…More precisely, no place shows a higher structural similarity than the site to which AB9 binds, although at the moment, we do not know where this site is. We do know, however, this site is a noncompetitive one [8] . It is highly likely that the "footprint" of AB9 site covers a larger surface area, which is needed to inhibit more selectively AMPA receptors.…”

“…Expressing a single target, i.e., one protein or receptor, to maximize the surface expression and density as opposed to expressing multiple receptors with "diluted" surface density for any one of the targets would be an advantage for this approach. In a recent article in the Journal of Biological Chemistry by Jaremko et al 2017 [8] , we have reported such an approach for the discovery and molecular characterization of an RNA aptamer that has a length-dependent, dual functionality against AMPA and kainate receptors.…”

“…Using our design approach and SELEX, we successfully isolated an aptamer, which we termed as "AB9" aptamer (101 nt), and a series of truncations of the full-length RNA led to a 55-nt RNA, which we termed as "AB9s" (Figure 1) [8] . There are several interesting features about AB9 and its short version AB9s.…”

“…Whole-cell current recording was used to measure the whole-cell current amplitude in the absence, A, and presence of an aptamer, A(I) (in these bar graphs, 2 µM aptamer was used in each assay). The potency and the selectivity of an aptamer against the open-channel, and the closed-channel state are assayed [8] .…”