GammaPNA Miniprobes for Telomere FISH

描述(由申请人提供)：拟议的第一阶段项目将把卡内基梅隆大学和匹兹堡大学开发的GammaPNA微型探头技术转让给位于匹兹堡的小型企业PNA InnoInnovation，Inc.。GammaPNA微型探针的基础是GammaPNA与互补DNA杂交的高亲和力。本提案中涉及的具体应用是端粒分析，这目前是使用长度为18个碱基的荧光PNA探针来完成的，该探针与人端粒序列5‘-AATGGG-3’的3个连续重复序列杂交。GammaPNA的高亲和力允许使用较短的12个碱基探针，导致更多的荧光染料被传递到给定长度的端粒。这将使对最短(即临界短)端粒的分析更加可靠，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12聚体微型探针，以最大限度地减少有效标记端粒所需的伽马修饰单体的数量。这将有助于将迷你探头的成本降低到商业PNA探头成本的大约一半(Panagene Inc.约1000美元的探头)。第二个目标是开发FRET对微型探头。微型探头的短长度将转化为高效的FRET。这一目的的意义将是消除在成像之前去除未杂交探针的洗涤步骤，因为FRET将仅在杂交探针之间发生。最终目标将导致开发在多个内部位置附着染料的微型探头。这将进一步增加微型探针的亮度，最高可达当前荧光PNA端粒探针的6倍。伽马PNA单体和低聚物的合成将在PNA创新公司进行。生物物理特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，伽马PNA微型探针技术就是在那里发明的。然后，优化型微型探针将被送往四个独立的贝塔测试实验室，这些实验室目前使用传统的PNA探针进行端粒分析。

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.