Telomeres are special sequences of DNA that occur at the 3' ends of chromosomes. They are responsible for anchoring the chromosome to the nuclear membrane and for protecting the DNA from exonucleolytic degradation. Reduction of telomere length has been observed with increasing number of cell divisions and a correlation has been found between telomere loss and cellular senescence. These observations have led to the telomere hypothesis which states that telomere upkeep is required for continued cell proliferation, while loss of telomere length leads to cell death. A unique DNA polymerase, telomerase, is a ribonucleoprotein that uses its RNA component as a template for synthesis of telomeric DNA. Consistent with the telomere hypothesis, normal somatic cells have little or no telomerase activity, while germline cells and most tumor cell lines have telomerase activity. To study the mechanism of telomerase, molecular probes will be prepared that are designed to bind to the RNA template of the enzyme. Peptide nucleic acid (PNA) will serve as the basic structural unit of the molecular probes. PNA is made up of an peptide-like backbone to which the normal nucleobases have been added. PNA binds tightly to DNA and RNA through normal Watson-Crick base pairing, and can be designed to target the RNA template of telomerase. Conjugates of PNA with other molecules such as peptides will be prepared in order to determine the accessibility of various regions of the RNA template. The ability of these molecules to inhibit telomerase will be determined using sensitive, quantitative assays such as the telomeric repeat amplification protocol (TRAP). In order to determine whether a correlation exists between affinity for telomeric oligonucleotides and inhibition of telomerase, PNA-peptide conjugates will be characterized for binding to oligonucleotides that mimic the RNA-DNA hybrid that is formed when telomerase synthesizes DNA. The peptides will be varied in sequence to present a variety of chemical functionality such as cationic, anionic, and neutral polymers to the Telomerase binding site. These studies will reveal new information regarding the mechanism of this important enzyme. Additionally, these compounds will be used to test the telomere hypothesis in human culture, and to assess the role of telomerase in telomere maintenance. Molecular probes for telomerase will be important new tools which will be useful to the study of the biology of telomeric DNA and its relationship to aging.

R03AG016103

1998-06-01

1999-05-31