CHEMICAL AND BIOLOGICAL MIMICRY OF TELOMERASE

描述(申请人提供)：我们实验室开发了一种新的仿生方法来高效合成端粒DNA。我们建议将这种方法应用于体外和细胞环境中端粒化学和生物学的研究。这种新的方法包括使用小的环状合成DNA作为模板，使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。通过仿生方法获得的端粒延长比用端粒酶本身实现的效率高得多，端粒酶很难或不可能以高活性分离出来。 特定的合成DNA环将被用来在体外产生人造人类端粒，其长度与自然端粒相似。该方法将用于制备单链和双链端粒，并将检测其结构、生物物理性质和蛋白质相互作用。此外，我们建议在完整的人类细胞中应用端粒延长方法，在没有该酶表达的情况下模拟体内端粒酶的作用。 在这项建议所涵盖的短期内，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)研究单链和双链形式的端粒聚合物的折叠；以及(Iii)开展针对活的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对端粒结构和功能的重要新的基本理解，为衰老和肿瘤发生提供有用的见解。应用于细胞，它可能导致新的方法来延长人类组织在培养中的有效生长寿命，在生物医学研究中有许多用途。化学方法延长端粒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疗法。

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.