Fluorescent Aptamers for Glycoprotein Detection

描述(由申请人提供)： 早期发现有助于提高癌症患者的存活率。实现这一目标的一种方法是检测和分析与癌症发展和预后相关的分子特征或生物标记物。沿着这条线，有必要开发各种癌症标记物的分子分析新技术。这种精神体现在RFA(RFA-CA-05-002)中，该文件要求应用程序开发新的“癌症检测技术和传感器，以及对癌症的发展和诊断至关重要的结构和分子”等。针对这种RFA，我们提出了一种新的平台技术的可行性研究，该技术可以用于快速构建糖蛋白荧光传感器。我们把重点放在糖蛋白上，因为许多这样的蛋白都与癌症的发展有关。该方法基于(1)通过指数富集法(SELEX)系统进化配体的能力，以寻找能够提供高亲和力和特异性识别目标分析物的最佳寡核苷酸适配子，(2)硼酸识别糖蛋白糖基部分上存在的二醇结构的独特能力，(3)我们自己开发的几种荧光硼酸化合物，它们在糖或糖蛋白结合时显示出非常显著的荧光强度变化(17-200倍)。我们希望在SELEX方法和硼酸对糖蛋白的独特识别之间建立协同作用，使基于DNA适体的荧光传感器(1)对目标糖蛋白具有高亲和力和特异性，(2)结合时显示非常显著的荧光强度变化。具体地说，该项目打算开发一种方法来制备用我们的荧光硼酸报告化合物修饰的DNA适配子。这些项目的具体目标包括：(1)合成与糖结合后表现出巨大荧光变化的荧光硼酸化合物；(2)将荧光硼部分引入核苷酸；3)使用SELEX方法选择具有最佳特异性和亲和力的传感器；4)验证与溶液中糖蛋白结合的传感器。对于这项可行性研究(R21)，我们选择前列腺特异性抗原(PSA)作为我们的模型糖蛋白，因为它在癌症诊断中的重要性，以及糖基化变化区分生理和病理PSA亚型的事实。这种荧光传感器如果被开发出来，将提供快速和灵敏的检测、高通量筛选的潜力和低成本的优势。此外，一旦开发出同样的技术，也可以用于构建其他与癌症相关的糖蛋白的荧光传感器。

DESCRIPTION (provided by applicant): Early detection helps to increase the survival rate in cancer patients. One way to achieve this is the detection and analysis of molecular signatures or biomarkers that have been correlated to cancer development and prognosis. Along this line, there is a need for the development of new technologies for the molecular analysis of various cancer markers. Such spirit is reflected in an RFA (RFA-CA-05-002) requesting applications on developing new "detection technologies and sensors of cancer and the structures and molecules important in its development and diagnosis," among other things. In response to this RFA, we propose this feasibility study of a new platform technology that can be used for the rapid construction of fluorescent sensors for glycoproteins. We focus on glycoproteins because numerous such proteins have been implicated in cancer development. This method is based upon (1) the power of systematic evolution of ligands by exponential enrichment method (SELEX) in search of optimal oligonucleotide aptamers that can afford high affinity and specificity recognition of the target analytes, (2) the unique ability of boronic acids to recognize diol structures present on the saccharide part of glycoproteins, (3) our own development of several fluorescent boronic acid compounds that show very significant fluorescence intensity changes (17- to 200-fold) upon saccharide or glycoprotein binding. We hope to build synergy between the SELEX approach and the unique recognition of glycoprotein by boronic acids in making DNA aptamer-based fluorescent sensors that (1) have high affinity and specificity for the target glycoprotein and (2) exhibit very significant fluorescence intensity changes upon binding. Specifically, the project intends to develop a method to prepare DNA aptamers modified with our fluorescent boronic acid reporter compounds. The specific aims of the projects include (1) the synthesis of fluorescent boronic acid compounds that show great fluorescence changes upon binding to saccharides, (2) incorporation of the fluorescent boronic moieties into nucleotides, 3) using the SELEX approach for the selection of sensors with optimal specificity and affinity, 4) validation of the sensor binding with glycoproteins in solution. For this feasibility study (R21), we have selected prostate-specific antigen (PSA) as our model glycoprotein because of its importance in cancer diagnosis and the fact that glycosylation variations distinguish between physiological and pathological PSA isoforms. Such fluorescent sensors, if developed, offer the advantage of rapid and sensitive detection, the potential for high throughput screening, and low cost. Furthermore, the same technology, once developed, can also be used for the construction of fluorescent sensors for other cancer-related glycoproteins.