CHEMICAL AND BIOLOGICAL MIMICRY OF TELOMERASE

描述（由申请人提供）：我们的实验室开发了一种新的仿生方法，用于有效合成端粒DNA。我们建议将这种方法应用于端粒化学和生物学的研究在体外和细胞的情况下。这种新方法涉及使用小的环状合成DNA作为模板，使得以前难以或不可能构建的聚合DNA结构和序列的制备成为可能。通过仿生方法实现的端粒延长比用端粒酶本身实现的端粒延长有效得多，端粒酶本身难以或不可能以高活性分离。 将在体外使用特定的合成DNA环组来产生长度与天然端粒相似的长聚合重复序列中的人工人类端粒。这种方法将用于制备单链和双链端粒，并将检查其结构，生物物理特性和蛋白质相互作用。此外，我们建议在完整的人类细胞中应用端粒延长方法，在体内模拟端粒酶的作用而不表达该酶。 在短期内，我们的具体目标是（i）测试环结构，大小和序列对体外端粒延长的影响;（ii）研究端粒聚合物在单链和双链形式下的折叠;以及（iii）进行针对活真核细胞中端粒延长的研究。 从长远来看，这种端粒酶模拟策略可以产生重要的端粒结构和功能的新的基本理解，对衰老和肿瘤发生提供有用的见解。应用于细胞，它可能会导致新的方法来延长培养中人体组织的有效生长寿命，在生物医学研究中有许多用途。端粒延长的化学方法具有解决移植医学中重要问题的长期潜力，并导致衰老相关疾病的新治疗方法。

DESCRIPTION (provided by applicant): 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.