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

 描述（由申请人提供）：我们实验室的长期目标是确定bPNA的结构-功能范围。本申请的目的是使用bPNA来产生荧光适体，其用作用于研究和治疗HER扩增的癌症的治疗诊断工具。本申请的中心假设是bPNA与核酸文库的杂交可用于将荧光团-光敏剂整合到亲和选择中，使得荧光团是与ERBB受体紧密接触的识别位点的组分。因此，靶结合激活荧光并使适体位点暴露于荧光体产生的活性氧物质的最大损伤。我们对这一假设的基本原理是发现bPNA三链体杂交可以荧光标记DNA和RNA适体。我们的独立努力得到了俄亥俄州州立大学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。这些因素联合收割机共同创造了一个有利于成功完成拟议调查的环境。拟议的研究是创造性和原创性的，因为一个新的通用方法描述了使用bPNA混合结构，将无缝地将非天然基团（荧光团）的亲和力选择。这将允许在适体-靶标识别界面选择人工官能团。bPNA-适体杂合结构提供了先前未知的变革优势。这种创造性的原始方法将产生以下预期结果：1）用于在SELEX中包含辅基的广泛适用的程序; 2）HER 2、HER 3和异二聚体状态的荧光报告物，以及报告任何适体位点的一般方案; 3）体外和细胞培养物中HER 2和HER 3的适体定向失活。拟议的研究将产生用于诊断和治疗HER扩增乳腺癌的治疗诊断荧光适体，并建立适体指导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.