GammaPNA Miniprobes for Telomere Analysis and RNA FISH

用于端粒分析和 RNA FISH 的 GammaPNA 微型探针

• 批准号: 9282753
• 负责人: Bruce A. ARMITAGE
• 金额: $ 33.22万
• 依托单位: PNA INNOVATIONS, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9282753
• 关键词: Address; Affinity; Age; Aging; Archives; Behavioral Research; Biological Assay; Biological Markers; Biological Sciences; Biology; Biomedical Research; Businesses; Cell Line; Cell Nucleus; Cell Proliferation; Cell division; Cells; Centromere; Chemistry; Chromosomal Stability; Chromosomes; Clinical; Color; Complementary DNA; DNA; DNA Damage; Data; Detection; Development; Diagnostic; Diagnostic tests; Diagnostics Research; Disease; Exhibits; Feedback; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Fluorescent Probes; Fluorescent in Situ Hybridization; Functional disorder; Generations; Goals; Hand; Health; Human; Hybrids; Inherited; Interphase; Jurkat Cells; Label; Laboratories; Left; Length; Libraries; Longevity; Malignant Neoplasms; Marketing; Messenger RNA; Methods; Modification; Molecular; Natural regeneration; Paper; Pathology; Peptide Nucleic Acids; Performance; Phase; Proteins; Publications; Publishing; RNA; RNA Sequences; RNA analysis; RNA library; Research; Research Personnel; Resolution; Sales; Sampling; Signal Transduction; Site; Solubility; Staining method; Stains; Technology; Telomerase; Telomere Maintenance; Testing; Tissue Sample; Tissues; Transgenic Mice; Universities; Vertebral column; Work; base; biophysical properties; cancer risk; cost; design; disorder risk; epidemiology study; experimental study; follow-up; human disease; improved; innovation; interest; member; monomer; peptide analog; public health relevance; response; success; telomere; tool

 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.

 描述（由适用提供）：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伽马普纳微型机器技术，随后转移到PNA Innovations，Inc，一家来自卡内基·梅隆大学的小型企业。伽马布纳小螺旋体的基础是伽马布纳杂交以完成DNA的高亲和力。本提案中涉及的特定应用是端粒分析，目前使用荧光PNA问题长度为18个基础，该碱基的长度为18个碱基，与人端粒序列5'-AATGGG-3'连续3次重复，并通过完整的荧光探针进行mRNA标记。在第一阶段，我们证明了γ的较高亲和力允许使用较短的12个基本端粒问题，从而导致更多的荧光染料传递到给定长度的端粒。这允许对最短的更可靠的分析（即批判性分析 简短）端粒，这些端粒在各种疾病中实施，包括与衰老有关的疾病和癌症。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结果，并围绕电视迷你游戏发起了一项营销活动，从而导致了我们在该产品系列中的首次销售。拟议的研究将具有四个具体目标。第一个目标以三种方式扩展了我们的阶段。首先，我们将研究其他几个细胞系，这将帮助我们（a）确定微型螺旋杆性能的变异范围，以及（b）潜在地识别在各种细胞系中起作用的微型机体的其他版本。其次，我们将开发针对富含C的端粒链的微型螺旋杆。第三，我们将基于承诺的初步结果，以综合内部标记的小型鞋，这将使我们当前最佳探针的亮度增加一倍或三倍。第二和第三目标是针对新应用的，特别是基于我们的微型生物和新鲜和存档的组织样品的高吞吐量端粒测定法，目前由于自动荧光而难以通过鱼类研究。第四个目标将显着将γ鱼探针扩展到RNA标记中。我们将合成针对单个mRNA不同地点的一组伽马广播，但我们将使用一种昂贵的探针标记探针，而是使用一种创新和经济的共同杂交方法来标记我们的问题。 PNA创新将完成Gammapna单体和低聚物的合成。生物物理特征和端粒染色将在卡内基·梅隆（Carnegie Mellon）和匹兹堡大学（University of Pittsburgh）的学术实验室中进行，并在那里发明了伽马普纳（Gammapna）微型机构技术。然后，优化的微型型将被发送到独立的β测试实验室，这些实验室目前使用常规的PNA问题进行端粒分析或基于DNA的分子信标进行mRNA标记。