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（由申请人提供）：拟议的第二阶段项目将继续开发最初由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 Innovations, Inc，这是一家从卡内基梅隆大学分离出来的小型企业。 gammaPNA 微型探针的优势在于 gammaPNA 与互补 DNA 杂交的高亲和力。该提案中提出的具体应用是端粒分析，目前使用荧光进行。 PNA 探针长度为 18 个碱基，与人类端粒序列 5'-AATGGG-3' 的 3 个连续重复序列杂交，并通过互补荧光探针进行 mRNA 标记。在第一阶段，我们证明 gammaPNA 的更高亲和力允许更短的 12 个碱基。使用端粒探针，导致更多的荧光染料被传递到给定长度的端粒，这使得对最短端粒（即关键端粒）的分析更加可靠。 简而言之）端粒，它与多种疾病有关，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研究结果，并围绕我们的 Telo MiniprobesTM 开展了营销活动，从而实现了我们的首次销售。拟议的研究将有四个具体目标。第一个目标以三种方式扩展我们的第一阶段工作。这将帮助我们（a）确定微型探针的变异范围。其次，我们将开发针对富含 C 的端粒链的微型探针；第三，我们将基于合成内部标记微型探针的有希望的初步结果。第二个和第三个目标是新的应用，特别是基于我们的微型探针的高通量端粒测定以及新鲜和存档组织的测试。由于自发荧光，目前很难通过 FISH 进行研究。第四个目标是将 gammaPNA FISH 探针显着扩展到 RNA 标记。我们将合成针对单个 mRNA 上不同位点的 gammaPNA 组，而不是共价标记探针。虽然成本高昂，但我们将使用创新且经济的共杂交方法来标记我们的探针。gammaPNA 单体和寡聚物的合成将在 PNA 生物物理表征中完成。端粒染色将在发明了 gammaPNA 微型探针技术的卡内基梅隆大学和匹兹堡大学的学术实验室进行，然后将优化的微型探针发送到目前使用传统 PNA 探针进行端粒分析或基于 DNA 的独立 β 测试实验室。用于 mRNA 标记的分子信标。