GammaPNA Miniprobes for Telomere FISH

用于端粒 FISH 的 GammaPNA 微型探针

• 批准号: 8591621
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8591621
• 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; 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把卡耐基梅隆大学和匹兹堡大学开发的gammaPNA微型探针技术转让给位于匹兹堡的一家小型企业PNA创新公司。gammaPNA微型探针的基础是gammaPNA与互补DNA杂交的高亲和力。本提案中提到的具体应用是端粒分析，目前使用长度为18个碱基的荧光PNA探针，杂交到人类端粒序列5‘- aatggg - 3’的连续3个重复。gammaPNA的高亲和力允许使用更短的12碱基探针，从而导致更多的荧光染料被传递到给定长度的端粒。这将允许对最短（即极短）端粒进行更可靠的分析，这些端粒与包括衰老相关疾病和癌症在内的各种疾病有关。拟议的研究将有三个具体目标。第一个目标是优化现有的12mer微型探针，以减少有效端粒标记所需的γ修饰单体的数量。这将有助于将微型探针的成本降低到商用PNA探针成本的大约一半（来自Panagene Inc .的约1000美元探针）。第二个目标是针对FRET对微型探针的发展。微型探针的短长度将转化为高效的FRET。这一目标的意义在于消除成像前去除未杂交探针的清洗步骤，因为FRET只会发生在杂交探针之间。最终目标将导致在多个内部位置附着染料的微型探针的发展。这将进一步提高微型探针的亮度，比目前的荧光PNA端粒探针高6倍。gammaPNA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耐基梅隆大学和匹兹堡大学的学术实验室进行，在那里发明了gammaPNA微型探针技术。然后，优化的微型探针将被送往四个独立的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。