Abstract
Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia. Glucagon exerts its physiological functions through activation of the glucagon receptor (GCGR). Inhibition of GCGR activity represents a potential therapeutic approach for reducing excess glucose production in diabetes mellitus. Aptamers are short DNA or RNA oligonucleotides evolved from systematic evolution of ligands by exponential enrichment (SELEX). Here, we have successfully selected a DNA aptamer against GCGR by cell-SELEX, which can specifically bind membrane protein of CHO-GCGR cells with a Kd of 52.7 ± 5.1 nM. Aptamer-mediated pull-down and gcgr knockdown assay verified that GCGR was the target of aptamer GR-3. Binding analysis revealed that GR-3 could recognize other cells with different affinity according to the level of GCGR protein expressed in these cells. Hepatic tissue imaging suggested that GR-3 could bind the cell membrane of hepatic tissues. With the advantages of small size, high binding affinity, good stability, lack of immunogenicity, and easy synthesis, aptamer GR-3 against GCGR can be a promising tool with the potential to attenuate hyperglycemia in diabetes mellitus.
Highlights
Glucagon, a 29-amino acid peptide secreted from pancreatic α cells, is a pivotal counter-regulatory hormone in the regulation of glucose homeostasis[1]
While red fluorescence was visualized on the cytomembrane of CHO-glucagon receptor (GCGR), almost no fluorescence was detected in Mock cells (Supplementary Fig. S1B), indicating high GCGR cell-surface expression in the CHO-GCGR cells
A DNA aptamer against GCGR has been successfully selected by cell-systematic evolution of ligands by exponential enrichment (SELEX) after 16 rounds of evolved enrichment
Summary
A 29-amino acid peptide secreted from pancreatic α cells, is a pivotal counter-regulatory hormone in the regulation of glucose homeostasis[1]. Aptamers are short DNA or RNA oligonucleotides evolved from random oligonucleotide libraries by a process called systematic evolution of ligands by exponential enrichment (SELEX)[18, 19] They can act as ligands with specific and high binding affinity for a variety of targets, including small molecules, proteins, nucleic acids, viruses, bacteria, cells and tissues[20, 21]. Some aptamers are able to retain their function to regulate biological pathways and interfere with disease development through binding to molecular targets involved in pathogenesis[24] Based on these advantages, aptamers show high potential for therapeutic applications, such as targeted therapy, detection and diagnostics[25,26,27,28]. A DNA aptamer against GCGR was developed by cell-SELEX, and its physical and biological properties were further investigated
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