GammaPNA Miniprobes for Telomere FISH

描述（由申请人提供）：拟议的第一阶段项目将转移gammaPNA微型探针技术开发的卡内基梅隆大学和匹兹堡大学的PNA创新公司，一个小企业位于匹兹堡。γ PNA微探针的基础是γ PNA与互补DNA杂交的高亲和力。本建议中提出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探针进行端粒分析，所述探针与人端粒序列5 ′-AATGGG-3 ′的3个连续重复序列杂交。γ PNA的较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递送到给定长度的端粒。这将允许更可靠地分析最短（即临界短）的端粒，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12聚体微探针，以最大限度地减少有效的端粒标记所需的γ-修饰的单体的数量。这将有助于将微型探针的成本降低到市售PNA探针的成本的大约一半（约100%）。Panagene Inc.提供的1000美元探测器）。第二个目标是针对FRET对的miniprobes的发展。小探针的短长度将转化为有效的FRET。该目的的重要性在于消除了在成像之前去除未杂交探针的洗涤步骤，因为FRET仅发生在杂交探针之间。最终的目标将导致开发具有在多个内部位置连接的染料的微型探针。这将进一步增加微探针的亮度，最多比当前荧光PNA端粒探针高6倍。γ PNA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内基梅隆大学和匹兹堡大学的学术实验室进行，那里发明了gammaPNA微型探针技术。然后，Optimize miniprobes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.