Our laboratory has developed a new biomimetic approach for efficient synthesis of telomeric DNAs. We propose to apply this methodology to the study of telomere chemistry and biology in vitro and in the cellular context. This new approach, which involves the use of small circular synthetic DNAs as templates, makes possible the preparation of polymeric DNA structures and sequences that were previously difficult or impossible to construct. The telomere elongation achieved by the biomimetic approach is far more efficient than can be achieved with telomerase itself, which is difficult or impossible to isolate with high activity. Specific sets of synthetic DNA circles will be used in vitro to generate artificial human telomeres in long polymeric repeats similar in length to natural telomeres. This method will be used to prepare single and double stranded telomeres, and their structure, biophysical properties, and protein interactions will be examined. Further, we propose to apply the telomere elongation approach in intact human cells, to mimic the action of telomerase in vivo without expression of the enzyme. In the short term covered by this proposal, our specific aims are (i) to test the effects of circle structure, size, and sequence on in vitro telomere elongation; (ii) to study folding of telomeric polymers in both single stranded and double stranded forms; and (iii) to carry out studies directed toward elongation of telomeres in living eukaryotic cells. In the long term, this telomerase mimetic strategy could generate important new basic understanding of telomere structure and function, giving useful insights into aging and oncogenesis. Applied in cells, it could result in new approaches to extending the effective growth lifespan of human tissue in culture, with many uses in biomedical research. Chemical approaches to telomere elongation have the long-term potential to solve important problems in transplantation medicine, and to lead to new treatments for senescence-related diseases.
| Pomerantz, Andrea K; Moerner, W E; Kool, Eric T (2008) Visualization of long human telomere mimics by single-molecule fluorescence imaging. J Phys Chem B 112:13184-7 |
| Hartig, Jorg S; Fernandez-Lopez, Sara; Kool, Eric T (2005) Guanine-rich DNA nanocircles for the synthesis and characterization of long cytosine-rich telomeric DNAs. Chembiochem 6:1458-62 |
| Hartig, Jorg S; Kool, Eric T (2005) Efficient isothermal expansion of human telomeric and minisatellite repeats by Thermococcus litoralis DNA polymerase. Nucleic Acids Res 33:4922-7 |
| Hartig, Jorg S; Kool, Eric T (2004) Small circular DNAs for synthesis of the human telomere repeat: varied sizes, structures and telomere-encoding activities. Nucleic Acids Res 32:e152 |
