GammaPNA Miniprobes for Telomere Analysis and RNA FISH

 描述（由申请人提供）：拟议的第二阶段项目将继续开发最初在卡内基梅隆大学和匹兹堡大学开发的gammaPNA微型探针技术，随后转移到PNA创新公司，一家从卡内基梅隆大学分拆出来的小企业。γ PNA微探针的基础是γ PNA与互补DNA杂交的高亲和力。在该提议中提出的具体应用是端粒分析，其目前使用长度为18个碱基的荧光PNA探针进行，所述荧光PNA探针与人端粒序列5 ′-AATGGG-3 ′的3个连续重复杂交，以及通过互补荧光探针标记mRNA。在第一阶段中，我们证明了γ PNA的较高亲和力允许使用较短的12个碱基的端粒探针，导致更多的荧光染料被递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） 短）端粒，其与包括衰老相关的疾病和癌症在内的多种病症有关。我们在《有机和生物分子化学》杂志上发表了一篇论文，描述了我们的研究结果，并围绕Telo MiniprobesTM开展了一场营销活动，从而实现了该产品线的首次销售。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第一阶段的工作。首先，我们将研究几种额外的细胞系。这将帮助我们（a）确定微探针性能的可变性范围和（B）潜在地鉴定在不同细胞系中工作的微探针的其他版本。其次，我们将开发针对富含C的端粒链的微型探针。第三，我们将建立在有希望的初步结果，合成内部标记的微型探针，这将是我们目前最好的探针的亮度的两倍或三倍。第二个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探针的高通量端粒测定和测试新鲜和存档的组织样品，这是目前难以通过FISH研究由于自发荧光。第四个目标是将γ PNA FISH探针显著扩展到RNA标记。我们将合成针对单个mRNA上不同位点的γ PNA组，但不是共价标记探针，这是昂贵的，我们将使用创新和经济的共杂交方法来标记我们的探针。γ PNA单体和低聚物的合成将在PNA Innovations完成。生物物理表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，那里发明了gammaPNA微型探针技术。然后，优化的微型探针将被送到独立的β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.