Selection and Characterization of Novel of RNA Aptamer Recognizing Homocysteine which Ameliorates Oxidative Stress in Mouse Brain Endothelial (bEND3) Cells

Abstract

Neurodegenerative diseases are characterized by a progressive deterioration of brain function, with a significant consequent decrease in the quality of patient's lives. Recently pathological mechanisms operating in cerebrovascular diseases mediated via homocysteine (Hcy) have gained increased attention. It has also been known that Hcy causes endothelial dysfunction by inducing oxidative stress in most cerebrovascular disorders. This dysfunction is highly correlated with reactive oxygen generation (ROS). Therefore, it is crucial to develop potential novel measures to slow down or prevent the onset of these diseases mediated by increased level of Hcy. Therefore, we proposed to select and characterize novel RNA aptamer recognizing Hcy to inhibit and modulates the Hcy activities involved in cerebrovascular disease. RNA Aptamers are specific bio‐molecules selected by the process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Aptamer folds into 3‐dimensional shapes which are capable of binding non‐covalently with high affinity and specificity to their target molecule. We have selected and characterized Hcy RNA aptamers using SELEX technology. The structural characterization was done by bioinformatics tools such as Clustal W, Mfold, IFold and HADDOCK. Binding characterization were done by CD and RNA EMSA studies. The best Hcy RNA aptamers were further investigated for their biological and functional characterization. Our results demonstrate that the Hcy RNA aptamer undergoes a conformation change upon ligand binding. These aptamers exhibit high specificity to ligand over nonspecific competitor. In our studies, bEND3 cells were exposed to Hcy treatment in the presence or absence of Hcy RNA aptamers for 24 hrs. We also demonstrated that these aptamers are novel class of RNA aptamer which ameliorates the effects of ROS thereby reducing the oxidative stress in bEND cell lines. Apoptotic and autophagic cell death were measured with 3‐(4, 5‐Dimethylthiazol‐2‐yl)‐2, 5‐ Diphenyltetrazolium Bromide (MTT) and monodansylcadaverine (MDC) staining assay. ROS production was determined using fluorescent probes 2, 7‐dichlorofluorescein acetate (DCFH‐DA. Autophagy flux was assessed using a conventional GFP‐microtubule‐associated protein 1 light chain 3 (LC3) dot assay. Mitochondrial fusion and fission was evaluated using Mfn‐2 and Drp‐1 RT‐PCR. Treatment of bEnd3 with Hcy RNA aptamer attenuated the production of free radicals and protected the cells from oxidative damage. Altogether, our studies strongly suggest that Hcy‐RNA aptamer has beneficial effects on cerebrovascular diseases and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy).Support or Funding InformationFinancial support from National Institute of health grant AR‐067667 and HL‐107640 are greatly acknowledged.