RNA and DNA Spiegelmers Binding to GnRH

More biological data for mirror-image oligonucleotides were generated by evaluating the properties of GnRH (gonadotropin-releasing hormone) binding spiegelmers [23]. GnRH is a ten-residue peptide released from the hypothalamus which acts as a key hormone of mammalian reproduction. The hormone binds to a single class of high affinity G-protein-coupled receptors on the plasma membranes of pituitary gonadotrophs and regulates the secretion of LH (luteinizing hormone) and FSH (follicle stimulating hormone). Inhibition of the GnRH-GnRH receptor system seems to be an appropriate form of treatment for certain sex hormone-dependent cancer indications such as prostate or breast cancer.

RNA and DNA libraries containing each 60 random positions and approx. 1015 different sequences were generated to isolate both RNA and DNA aptamers against D-GnRH. For partitioning, the oligonucleotide libraries were applied to sepharose columns which were derivatized with modified D-GnRH-Cys. The binding oligonucleotides were eluted by affinity competition with selection buffer containing unmodified D-GnRH. After six and eight rounds of in-vitro selection for the RNA and DNA libraries, respectively, the enriched libraries were cloned and sequenced. The sequences of the enriched RNA library were dominated by one main sequence and some related sequences which differed only in point mutations. The KD of the main RNA sequence was found to be 92 + 12 ra. The enriched DNA library revealed three clones that occurred between 10 and 15 times, as well as several orphan sequences. One of the orphans had the best KD, 55 + 7 ra, and was therefore chosen for further analysis. The RNA sequence was readily truncated to a 50-mer (see Appendix). Both RNA and mirror-image RNA had binding properties similar to those of their respective targets, although the dissociation constants rose slightly to 190-260 nM as a result of the truncation. The DNA could be truncated and modified to a 60-mer (see Appendix); both DNA and mirror-image DNA bound to GnRH with equilibrium dissociation constants of approximately 46 + 7 nM (Figure 3.4.6). The RNA and DNA spiegelmers showed specificity by not binding to other peptides, even if they were related to GnRH, like buserelin and chicken LHRH. The functional activity of the spiegelmers was shown in a cell based assay employing Chinese hamster ovary cells expressing the GnRH receptor. GnRH receptor binding results in intracellular Ca2+ release that can be detected by


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