Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul 16;5(7):559-67.
doi: 10.1021/cn500041k. Epub 2014 Apr 16.

RNA Based Antagonist of NMDA Receptors

Affiliations
Free PMC article

RNA Based Antagonist of NMDA Receptors

Garam Lee et al. ACS Chem Neurosci. .
Free PMC article

Abstract

The N-methyl d-aspartate (NMDA) class of ionotropic glutamate receptors plays important roles in learning and memory as well as in a number of neurological disorders including Huntington's disease and cerebral ischemia. Here, we describe the isolation and characterization of a 2' F-modified RNA aptamers directed against GluN2A-containing NMDA receptors. By adding a negative selection step toward the closely related AMPA and kainate receptors, the RNA aptamers specifically recognize NMDA receptors with dissociation constants in the nanomolar range. Electrophysiological characterization of these aptamers using rapid perfusion in outside-out patches reveals that they selectively inhibit the GluN2A containing subtype of NMDA receptors with little effect on the AMPA and kainate receptor subtypes. We also demonstrate that this RNA aptamer significantly reduces neurotoxicity in an in vitro model of cerebral ischemia. Given that the RNA based antagonist can be readily modified, it can be used as a tool in targeted drug delivery or for imaging purposes in addition to having the potential use as a therapeutic intervention in disorders involving glutamate receptors.

Figures

Figure 1
Figure 1
Radioligand binding curves before and after SELEX. (A) Binding curves for the starting pool (black circles) and the pool after 9 rounds of SELEX (gray triangles) on GluN1/GluN2A NMDA receptors. Note saturation binding in the round 9 pool. (B) Similar binding curves on GluA2 AMPA receptors. (C) Binding curves for GluK2 KA receptors.
Figure 2
Figure 2
Truncation of candidate C26 aptamer. (A) Predicted secondary structure of the C26 aptamer showing segments truncated. (B) Radioligand binding curves for the indicated aptamer on GluN1/GluN2A NMDA receptors. Note the loss of saturating binding in C26–31 aptamer corresponding to a reduction in binding affinity.
Figure 3
Figure 3
Glutamate receptor inhibition by truncated C26 aptamers. (A) Example responses to a 1 ms pulse of 10 mM glutamate in outside out patches containing GluN1/GluN2A NMDA receptors (upper row), GluA2 AMPA receptors (middle row), and GluK2 KA receptors (lower row) in the presence of 1 μM C26 (left column), C26–50 (middle column), and C26–31 (right column). Note NMDA receptor inhibition is reduced with C26–31. (B) Summary of inhibition by 1 μM of various truncated aptamers for each receptor type.
Figure 4
Figure 4
Inhibition curve of peak GluN1/GluN2A responses by the C26–50 aptamer. (A) Example responses of an outside-out patch containing GluN1/GluN2A receptors to a 2 s application of 10 mM glutamate alone or in the continual presence of the indicated concentration of C26–50 aptamer. (B) Summary of peak inhibition data.
Figure 5
Figure 5
C26–50 is a noncompetitive inhibitor. (A) Representative whole cell responses to 100 mM glutamate alone (black trace), coapplied with 1 mM C26–50 (gray trace) or 1 mM glutamate coapplied with C26–50 (dark gray trace). Note that increasing glutamate concentration 10-fold does not relieve steady-state inhibition. (B) Summary of competition experiment showing additional glutamate did not relieve C26–50 inhibition. (C) Glutamate dose–response curves in the absence (black circles) and presence (red circles) of 1 μM C26–50. (D) Schild plot showing the shift in glutamate EC50 in the presence of various concentrations of C26–50 aptamer. The slope of the fit is 0.6 ± 0.1, confirming that C26–50 is a noncompetitive inhibitor.
Figure 6
Figure 6
Neuronal injury and viability measured by LDH and MTT assays. (A) Percentages of neuronal survival in the presence of the indicated compounds without OGD. Note the absence of toxicity of these compounds. (B) Neuronal survival in the presence of the indicated compounds following OGD. Note that the C26–50 aptamer was neuroprotective at levels concentrations to memantine.

Similar articles

See all similar articles

Cited by 4 articles

Publication types

MeSH terms

Feedback