After incubation, the supernatant was discarded, and cells were washed with 3?mL of cleaning buffer to remove unbound sequences

After incubation, the supernatant was discarded, and cells were washed with 3?mL of cleaning buffer to remove unbound sequences. advantages Alofanib (RPT835) of Alofanib (RPT835) 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. Introduction Glucagon, a 29-amino acid peptide secreted from pancreatic cells, is a pivotal counter-regulatory hormone in the regulation of glucose homeostasis1. Glucagon stimulates hepatic glucose production and output by promoting glycogenolysis and gluconeogenesis (GNG) in the liver and attenuates the ability of insulin to inhibit these processes in the fasting state2. Glucagon exerts its physiological functions through activation of the glucagon receptor (GCGR), which is predominantly localized in the liver3, 4. GCGR is a seven trans-membrane G protein-coupled receptor consisting of 485 Alofanib (RPT835) amino acids. In patients with type 2 diabetes mellitus (T2DM), the secretion of glucagon is increased in both the fasting and postprandial states and contributes to pathogenesis of diabetic hyperglycemia through excessive hepatic glucose production and output5, 6. Recent studies revealed that excessive glucagon secretion or action, rather than insulin deficiency, is predominant in the progress of diabetes7, 8. Accordingly, inhibition of GCGR activity represents a potential therapeutic approach for reducing excess glucose production in patients with T2DM. For instance, reduction in GCGR expression using antisense oligonucleotides (ASOs) has been shown to lower glycemia and ameliorate metabolic syndrome in mice and Zucker diabetic fatty rats9C11. Extensive efforts have been undertaken by the pharmaceutical Alofanib (RPT835) industry to develop potent small molecule glucagon receptor antagonists or antibodies for clinical use12, 13. Several glucagon receptor antagonists and antibodies able to improve glucose homeostasis in animal models and humans have been reported14C16. However, thus far none has progressed to final marketing approval, mainly due to a poor performance profile, including toxicity or lack of selectivity17. 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 tissues20, 21. The molecular recognition properties of aptamers are similar to those of antibodies. However, manmade aptamers possess several advantages over naturally occurring antibodies, including economical and reproducible synthesis, easy modification, low toxicity, high stability, lack of immunogenicity, and rapid tissue penetration22, 23. In addition to recognition, some aptamers are able to retain their function to regulate biological pathways and interfere with disease development through binding to molecular targets involved in pathogenesis24. Based on these advantages, aptamers show high potential for therapeutic applications, such as targeted therapy, detection and diagnostics25C28. Macugen, the first aptamer-based drug approved by the U.S. Food and Drug Administration (FDA) in 2004, is now available for treatment of age-related macular degeneration (AMD)29. Other aptamers, such as aptamer AS1411, which is specific for nucleolin, are currently undergoing clinical evaluation30. This indicates that aptamers can also be used directly as drugs18. For the selection of anti-protein aptamers, PCDH8 SELEX is usually carried out using purified recombinant proteins. Therefore, the precondition of SELEX for anti-protein aptamers is the preparation of sufficient amounts of high-quality, purified protein31. However, many pharmacologically relevant cell membrane proteins, such as G protein-coupled receptors, cannot be purified because of their associated complexity and instability32. However, SELEX against live cells (cell-SELEX) has enabled the generation of aptamers which, with their flexible conformations, can specifically bind target molecules in their native state on the cell surface without prior knowledge of the molecular signatures of target cells18, 33. As such, target molecules do not require purification or anchorage on a solid support by processes that may Alofanib (RPT835) destroy their native conformations34. Additionally, some aptamers selected by cell-SELEX strategies are endowed with inhibitory activity by binding with.