Abstract
Ligands of high specificity and selectivity have been selected for biological molecules of interest including nicotinic acetylcholine receptor (nAChR) using combinatorial libraries of nucleic acids. The nAChR belongs to a group of structurally related proteins that regulate signal transmission between ~ 1012 cells of the mammalian nervous system. It is inhibited by both therapeutic agents and abused drugs, including cocaine. A mechanism-based approach to alleviating noncompetitive inhibition of the mucle-type nAChR, including Torpedo, resulted in the selection of very short DNA aptamers only seven nucleotides long. By transient kinetic measurements, these DNA aptamers, which displaced cocaine from its binding site on the muscle-type nAChR, were classified into two groups based on their effects on the nAChR: Class I aptamers inhibit agonist-induced current in the muscle-type nAChR and Class II molecules alleviate inhibition by MK-801 [(+)-dizocilpine] without affecting the receptor function. The most potent Class I DNA aptamer, which inhibits the muscle-type nAChR, has an apparent dissociation constant (KIapt) of 5 μM, while the most efficient Class II DNA aptamer, which alleviates MK-801-induced inhibition, has an apparent dissociation constant (KApt) of 1.8 μM. An innovative aspect of the work is the identification of very short DNA aptamers with these properties that makes them attractive for therapeutic and diagnostic applications.
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Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- nAChR:
-
Nicotinic acetylcholine receptor
- RNA:
-
Ribonucleic acid
- DNA:
-
Deoxyribonucleic acid
- SELEX:
-
Systematic evolution of ligands by exponential enrichment
- PCP:
-
Phencyclidine
- Nt:
-
Nucleotides
- dsDNA:
-
Double-stranded DNA
- ssDNA:
-
Single-stranded DNA
- Hepes:
-
N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- EDTA:
-
Ethylene diamine tetraacetic acid
- DNase:
-
Deoxyribonuclease
- Da:
-
Daltons
- MK-801:
-
(+)-Dizocilpine
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Acknowledgements
This work was supported by a National Institutes of Health grants awarded to George P. Hess, (NS08527). We thank Dr. Oné Pagán (West Chester University of Pennsylvania, West Chester, PA) for the help in preparing the T. californica membranes.
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This article is dedicated to the memory of late Professor George P. Hess.
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Sivaprakasam, K., Hess, G.P. In Vitro Selection of Short DNA Aptamers that Can Inhibit or Alleviate Cocaine and MK-801 Inhibition of Muscle-Type Nicotinic Acetylcholine Receptors. J Membrane Biol 255, 41–53 (2022). https://doi.org/10.1007/s00232-021-00202-0
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DOI: https://doi.org/10.1007/s00232-021-00202-0