VEGF, was approved by the FDA for treatment of neovascular age-related macular degeneration in 2004. Aptamers have previously been used to investigate neurological disorders, such as Alzheimer��s, multiple sclerosis, and myasthenia gravis. For example, an aptamer was selected against the 40 amino-acid beta-amyloid peptide and was shown to bind fibrils consisting of the peptide. But no functional data regarding fibril dissociation or reduction has been reported. Similarly, aptamers have been used to SB431542 target myasthenia gravis, which is a neuromuscular disorder resulting from antibodymediated autoimmune response to the nicotinic acetylcholine receptor. A 29-amino-modified aptamer was isolated against Mab198, a monoclonal antibody that recognizes the major immunogenic epitope on human AchR. The aptamer protected AChR from antoantibodies found in patients with myasthenia gravis. A later selection yielded a 29-fluoropyrimidinemodified aptamer, which offered even greater protection. However, in these instances aptamers have primarily 
been used to treat disorders, rather than to modulate normal neuronal function. Here, we selected RNA aptamers that bind to NgR with high specificity and affinity. Most importantly, these aptamers were shown to compete with Nogo, MAG, and OMgp for binding to NgR. Neurite outgrowth assays demonstrated that these aptamers can reverse the effect of these inhibitors in vitro. These are the first aptamers to modulate neuronal growth. A RNA pool containing 50 randomized positions flanked by two constant, primer-binding regions was used as a starting point for selection. The protein target was a fusion of the extracellular domain of rat NgR and the constant region of human IgG. In each round of selection, RNA-binding species were separated from weak or non-binding species by passing the protein:nucleic acid complexes through a modified nitrocellulose filter. Captured species were amplified via reverse transcription and PCR. Negative selections were GDC-0879 carried out to remove aptamers that bound species other than the target. Iterative rounds of selection and amplification were performed until the target-binding aptamers dominated the selected population. To further improve affinity, additional variants were selected from a partially randomized pool based on Clone 6 that contained 70% wild-type and 30% non-wild type nucleotides. This level of mutagenesis should allow the accumulation of not only additional functional residues, but also changes in or additions to base-paired structures. In addition, a new selection was carried out with a slightly longer pool in order to identify aptamers that might cover larger swaths of the NgR surface and thereby prevent binding of multiple inhibitors. At the conclusion of both selections, 56 clones were sequenced: 28 from the Clone 6 doped re-selection, and 28 from the N62 selection. As expected, the best ligands for NgR were present multiple times in the selected populations. However, no strong consensus sequences or motifs emerged from the selections. The diversity of aptamer sequences could mean that the selected aptamers interact in several different ways with NgR, a result that is consistent with the fact that very different myelinderived inhibitors all bind to NgR. The aptamers that were used for in vitro studies were chosen based both on affinity and on their ability to bind to non-overlapping sites. The identified aptamers bound NgR with Kd values from 21 to 61 nM. Competition assays between aptamers were carried out in order to determine whether they bound to the same, different, or overlapping epitopes.