IDENTIFICATION OF DETERMINANTS FOR INHIBITOR BINDING WITHIN THE RNA ACTIVE-SITE OF HUMAN TELOMERASE USING PNA SCANNING

Telomerase is a ribonucleoprotein that participates in the maintenance of telomere length. Its activity is up-regulated in many tumor types, suggesting that it may be a novel target for chemotherapy. The RNA co mponent of telomerase contains an active site that plays at least two roles-binding telomere ends and templating their replication [Greicer, C. W., & Blackburn, E. H, (1989) Nature 337, 331-337]. The accessibil ity of RNA nucleotides for inhibitor binding cannot be assumed because of the potential for RNA secondary structure and RNA-protein interact ions. Here we use high-affinity recognition by overlapping peptide nuc leic acids (PNAs) [Nielsen, P. E., et al. (1991) Science 254, 1497-150 0] to identify nucleotides within the RNA active site of telomerase th at are determinants for inhibitor recognition. The IC50 for inhibition decreases from 30 mu M to 10 nM as cytidines 50-52 (C50-52) at the bo undary between the alignment and elongation domains are recognized by PNAs overlapping from the 5' direction. As C50-52 are uncovered in the 3' direction, IC50 increases from 10 nM to 300 nM. As cytidine 56 at the extreme 3' end of the active site is uncovered, IC50 values increa se from 0.5 mu M to 10 mu M. This analysis demonstrates that C50-C52 a nd C56 are important for PNA recognition and are physically accessible for inhibitor binding. We use identification of these key determinant s to minimize the size of PNA inhibitors, and knowledge of these deter minants should facilitate design of other small molecules capable of t argeting telomerase. The striking differences in IC50 values for inhib ition of telomerase activity by related PNAs emphasize the potential o f PNAs to be sensitive probes for mapping complex nucleic acids. We al so find that PNA hybridization is sensitive to nearest-neighbor intera ctions, and that consecutive guanine bases within a PNA strand increas e binding to complementary DNA and RNA sequences.