GammaPNA Miniprobes for Telomere Analysis and RNA FISH

 描述(申请人提供)：拟议的第二阶段项目将继续开发GammaPNA微型探头技术，该技术最初由卡内基梅隆大学和匹兹堡大学开发，随后转移到PNA创新公司，该公司是从卡内基梅隆大学剥离出来的一家小企业。GammaPNA微型探针的基础是GammaPNA与互补DNA杂交的高亲和力。本提案中涉及的具体应用是端粒分析，这是目前使用长度为18个碱基的荧光PNA探针来完成的，该探针与人端粒序列5‘-AATGGG-3’的3个连续重复序列杂交，以及通过互补荧光探针标记mRNA。在第一阶段，我们证明了GammaPNA的高亲和力允许使用更短的12碱基端粒探针，导致更多的荧光染料被输送到给定长度的端粒。这允许更可靠地分析最短的(即关键的 简而言之，端粒与多种疾病有关，包括与衰老相关的疾病和癌症。我们发表了一篇论文，描述了我们在有机和生物分子化学方面的成果，并围绕我们的Telo微型探测器TM发起了一项营销活动，导致了我们在这一产品线内的首次销售。这项拟议的研究将有四个具体目标。第一个目标从三个方面扩展了我们的第一阶段工作。首先，我们将研究几个额外的细胞系，这将有助于我们(A)确定微型探针性能的可变性范围，以及(B)潜在地确定在不同细胞系中工作的其他版本的微型探针。其次，我们将开发针对富含C的端粒链的微型探针。第三，我们将在有希望的初步结果的基础上合成内部标记的微型探测器，这将使我们目前最好的探测器的亮度增加一倍或三倍。第二和第三个目标是针对新的应用，特别是在开发基于我们的微型探针的高通量端粒分析以及对新鲜和存档的组织样本的测试方面，这些组织样本目前由于自身荧光而难以通过FISH进行研究。第四个目标是将GammaPNA FISH探针显著扩展到RNA标记。我们将合成一组针对单个RNA上不同位点的GammaPNAs，但我们将使用一种创新和经济的共杂交方法来标记我们的探针，而不是共价标记探针。伽马PNA单体和低聚物的合成将在PNA创新公司进行。生物物理特征和端粒染色将在卡内基梅隆大学和匹兹堡大学的学术实验室进行，伽马PNA微型探针技术就是在那里发明的。然后，优化的微型探针将被送往独立的Beta测试实验室，这些实验室目前使用传统的PNA探针进行端粒分析，或使用基于DNA的分子信标进行mRNA标记。

 DESCRIPTION (provided by applicant): The proposed Phase II project will continue development of gammaPNA miniprobe technology originally developed at Carnegie Mellon University and the University of Pittsburgh and subsequently transferred to PNA Innovations, Inc, a small business spun out of Carnegie Mellon University. The basis of gammaPNA miniprobes is the high affinity with which gammaPNA hybridizes to complementary DNA. The specific applications addressed in this proposal are telomere analysis, which is currently done using fluorescent PNA probes 18 bases in length, which hybridize to 3 consecutive repeats of the human telomere sequence 5'-AATGGG-3', and mRNA labeling by complementary fluorescent probes. In Phase I, we demonstrated that the higher affinity of gammaPNA allows shorter 12 base telomere probes to be used, resulting in more fluorescent dyes being delivered to a telomere of a given length. This allow more reliable analysis of the shortest (i.e. critically short) telomeres, which are implicated in a variety of conditions including aging-related diseases and cancer. We published a paper describing our results in Organic and Biomolecular Chemistry and we launched a marketing campaign around our Telo MiniprobesTM, leading to our first sales within this product line. The proposed research will have four Specific Aims. The first aim extends our Phase I work in three ways. First we will study several additional cell lines This will help us to (a) determine the range of variability in miniprobe performance and (b) potentially identify other versions of the miniprobe that work in varied cell lines. Second, we wil develop miniprobes that target the C-rich telomere strand. Third, we will build on promising preliminary results for synthesizing internally labeled miniprobes that will double or triple the brightness of our current best probe. The second and third aims are directed toward new applications, specifically in development of a high throughput telomere assay based on our miniprobes and testing of fresh and archived tissue samples, which are currently difficult to study by FISH due to autofluorescence. The fourth aim will significantly extend gammaPNA FISH probes into RNA labeling. We will synthesize sets of gammaPNAs targeted to different sites on a single mRNA, but rather than covalently label the probes, which is costly, we will use an innovative and economical co-hybridization approach to label our probes. The synthesis of the gammaPNA monomers and oligomers will be done at PNA Innovations. Biophysical characterization and telomere staining will be done at academic laboratories at Carnegie Mellon and the University of Pittsburgh where the gammaPNA miniprobe technology was invented. Optimized miniprobes will then be sent to independent beta-testing laboratories that currently use conventional PNA probes for telomere analysis or DNA-based molecular beacons for mRNA labeling.