GammaPNA Miniprobes for Telomere FISH

用于端粒 FISH 的 GammaPNA 微型探针

• 批准号: 8728970
• 负责人: Bruce A. ARMITAGE
• 金额: $ 27.03万
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8728970
• 关键词: Address; Adenine; Affinity; Age; Base Pairing; Behavioral Research; Biological Assay; Biological Markers; Biomedical Research; Businesses; Cancerous; Cell Proliferation; Cell division; Chemistry; Chromosomal Stability; Chromosomes; Complementary DNA; Cytosine; DNA; Data; Detection; Development; Disease; Dyes; Energy Transfer; Exhibits; Feedback; Fluorescence; Fluorescent Dyes; Fluorescent in Situ Hybridization; Generations; Goals; Health; Human; Image; Individual; Inherited; Intervention; Label; Laboratories; Length; Lesion; Letters; Life Style; Longevity; Malignant Neoplasms; Modification; Natural regeneration; Noise; Nucleic Acid Probes; Pathology; Peptide Nucleic Acids; Performance; Phase; Positioning Attribute; Production; RNA; Reporting; Research; Risk; Signal Transduction; Solubility; Staining method; Stains; Stress; Technology; Testing; Thymine; Tissues; Transgenic Mice; Translating; Treatment Efficacy; Universities; Uracil; Vertebral column; Work; age related; analog; base; biophysical properties; cancer risk; cost; disorder risk; epidemiology study; human disease; improved; innovation; monomer; public health relevance; telomere; tool

DESCRIPTION (provided by applicant): The proposed Phase I project will transfer gammaPNA miniprobe technology developed at Carnegie Mellon University and the University of Pittsburgh to PNA Innovations, Inc, a small business located in Pittsburgh. The basis of gammaPNA miniprobes is the high affinity with which gammaPNA hybridizes to complementary DNA. The specific application addressed in this proposal is 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'. The higher affinity of gammaPNA allows shorter 12 base probes to be used, resulting in more fluorescent dyes being delivered to a telomere of a given length. This will allow more reliable analysis of the shortest (i.e. criticaly short) telomeres, which are implicated in a variety of conditions including aging-related diseases and cancer. The proposed research will have three specific aims. The first aim concerns optimization of an existing 12mer miniprobe to minimize the number of gamma-modified monomers needed for effective telomere labeling. This will help to drive down the cost of the miniprobe to approximately one half the cost of commercially available PNA probes (ca. $1000probe from Panagene Inc). The second aim is directed toward development of FRET pairs of miniprobes. The short length of the miniprobes will translate into efficient FRET. The significance of this aim will be the elimination of washing steps to remove unhybridized probes prior to imaging, since FRET will only occur between hybridized probes. The final aim will result in development of miniprobes having dyes attached at multiple internal positions. This will further increase the brightness of the miniprobes, up to a factor of 6 over current fluorescent PNA telomere 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. Optimize miniprobes will then be sent to four independent beta-testing laboratories that currently use conventional PNA probes for telomere analysis.

描述（由申请人提供）：拟议的第一阶段项目将转移卡内基·梅隆大学和匹兹堡大学开发的Gammapna Miniprobe技术，到位于匹兹堡的小型企业PNA Innovations，Inc。伽马布纳小螺旋体的基础是伽马布纳杂交与互补DNA的高亲和力。本提案中解决的特定应用是端粒分析，目前使用荧光PNA探针长度为18个基础，该碱基的长度与人类端粒序列5'-AATGGG-3的连续3个重复序列杂交。 Gammapna的较高亲和力允许使用12个基本探针，从而导致更多的荧光染料传递到给定长度的端粒。这将允许对最短（即关键短）端粒进行更可靠的分析，这些分析与各种疾病有关，包括与衰老有关的疾病和癌症。拟议的研究将具有三个具体目标。第一个目的是优化现有的12mer小型企业，以最大程度地减少有效端粒标签所需的伽马修饰单体数量。这将有助于将微型企业的成本降低到市售PNA探针成本的一半（panagene Inc的$ 1000Probe）。第二个目的是针对小型生物对开发。小型鞋的短长度将转化为有效的粮食。该目标的重要性将是消除在成像之前去除杂交探针的洗涤步骤，因为仅在杂交探针之间出现FRET。最终目标将导致在多个内部位置附着染料的微型鞋的发展。这将进一步提高微型鞋的亮度，超过当前荧光PNA端粒探针的6倍。 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，优化微型型将被发送到四个独立的β测试实验室，这些实验室目前使用常规的PNA探针进行端粒分析。