APTAMERS-BLOCKING PROTEINS WITH RNA
In theory, antisense oligonucleotides could also disrupt gene expression by preventing transcription factors from binding to DNA. Many DNA binding proteins have relatively short recognition sequences; therefore, short oligonucleotides could block the DNA binding site of the protein. Without is an active transcription factor, the target gene would not be transcribed.
In principle, an artificial oligonucleotide could be designed to fit into a binding site of any shape. Even if an enzyme does not normally bind nucleic acids, an oligonucleotide could be designed that would block the active site of the protein. Such oligonucleotides do not correspond to natural nucleic acid sequences and are known as aptamers. This procedure has been demonstrated experimentally for the enzyme thrombin, a protease involved in the blood clotting cascade. The antithrombin aptamer is short-lived because it is degraded rapidly in vivo. It might perhaps be useful as an anticlotting agent, such as during bypass surgery.
Aptamers may be made by a combination of molecular selection and PCR amplification. Random oligonucleotides (of 50 or 60 bases) are artificially synthesized by solid-state synthesis, using a mixture of all four bases at each step. The pool of random sequences is then ligated at each end to primer binding sequences (approximately 20 bases long). The mixture is poured through a column to which the target protein is attached (Fig. 17.14). Some oligonucleotides will bind to the protein and are therefore retained by the column. These are dissociated and then amplified by PCR. The bound oligonucleotides will be a mixture, some of which bind weakly and some strongly. Therefore the binding and PCR steps are repeated two or three times to select oligonucleotides with high binding affinity.
Recently, pegaptanib (Macugen; from Eyetech Pharmaceuticals, Pfizer, New York) has been approved for treatment of blindness due to age-related macular degeneration. Pegaptanib is an aptamer that inhibits the action of an isoform of vascular endothelial growth factor (VEGF) that is responsible for abnormal vascularization of the eyes. Binding of pegaptanib to VEGF prevents VEGF from binding to its receptor.
Copyright © 2018-2020 BrainKart.com; All Rights Reserved. Developed by Therithal info, Chennai.