Development of Fluorogenic Aptamers for Detection and Deactivation of Erbb Receptors using Bifacial PNA

 描述(申请人提供)：我们实验室的一个长期目标是确定bPNA的结构-功能范围。这项应用的目的是使用bPNA生产荧光适体，作为治疗工具用于研究和治疗HER扩增的癌症。这一应用的中心假设是，bPNA与核酸文库的杂交可以用于将荧光素-光敏剂整合到亲和选择中，从而使荧光素成为与ERBB受体密切接触的识别位点的组成部分。因此，靶标结合激活了荧光，并使配基部位受到荧光素产生的活性氧物种的最大损害。我们对这一假设的基本原理是发现bPNA三链杂交可以荧光标记DNA和RNA适配子。我们的独立努力得到了俄亥俄州立大学RNA生物学中心的共享资源和专业知识的支持，以及HER+乳腺癌临床研究、ERBB受体生物化学、SELEX、ERBB失调癌症的光免疫疗法和光物理方法的合作者的支持。这些因素加在一起，创造了一个有利于成功完成拟议调查的环境。这项拟议的研究具有创造性和原创性，因为使用bPNA杂交结构描述了一种新的通用方法，该结构将无缝地将非天然基团(荧光素)合并到亲和选择中。这将允许在适配子-靶识别界面选择人工官能团。BPNA-适配子杂交结构提供了以前未知的转化优势。这一创造性、独创性的方法将产生以下预期结果：1)SELEX中广泛适用的包括假体基团的程序；2)HER2、HER3和异二聚体状态的荧光报告，以及报告任何适配位点的通用程序；3)适体诱导的体外和细胞培养中HER2和HER3的失活。这项拟议的研究将产生用于诊断和治疗HER扩增的乳腺癌的声学荧光适体，并建立适体导向的生物传感器和光动力治疗试剂的通用方案。

 DESCRIPTION (provided by applicant): A long term goal in our lab is to determine the structure-function scope of bPNA. The objective of this application is to use bPNA to produce fluorogenic aptamers that are function as theranostic tools for study and treatment of HER-amplified cancer. The central hypothesis of this application is that bPNA hybridization with nucleic acid libraries can be used to integrate fluorogen-photosensitizers into affinity selection such that the fluorogen is a component of the recognition site that makes close contact with the ERBB receptor. Target binding thus activates fluorescence and exposes the aptagenic site to maximum damage by fluorogen-produced reactive oxygen species. Our rationale for this hypothesis is the finding that bPNA triplex hybridization can fluorescently label DNA and RNA aptamers. Our independent efforts are supported by the shared resources and expertise of the Center for RNA Biology at the Ohio State University as well as collaborators in clinical investigation of HER+ breast cancer, ERBB receptor biochemistry, SELEX, photoimmunotherapy on ERBB-dysregulated cancer and photo physical methods. These factors combine to create a setting conducive to the successful completion of the proposed investigations. The proposed research is creative and original because a new general method is described using bPNA hybrid structures that will seamlessly incorporate non-native groups (fluorogens) into affinity selection. This will allow selection for artificial functional groups atthe aptamer-target recognition interface. The bPNA-aptamer hybrid structures offer transformative advantages not previously known. This creative, original approach will yield the following expected outcomes: 1) a widely applicable procedure for inclusion of prosthetic groups in SELEX; 2) fluorogenic reporters of HER2, HER3 and heterodimer status, as well as a general protocol to report on any aptagenic site; 3) aptamer- directed inactivation of HER2 and HER3 in vitro and in cell culture. The proposed research will yield theranostic fluorogenic aptamers for diagnosis and treatment of HER-amplified breast cancer as well as establish a general protocol for aptamer-directed biosensors and photodynamic therapeutic reagents.