LPS-specific Aptamers for Microbial Detection

描述(申请人提供)：内毒素是革兰氏阴性细菌表面的一个显著特征。识别和区分不同O-特异性寡糖单位的抗体被用来识别在受污染的食品中发现的、导致人类死亡和疾病的致病细菌菌株。内毒素分子经常与致病机制密切相关，并参与细菌黏附和运动等功能，以及引发自身免疫反应。败血症休克综合征是一种严重的炎症反应，通常由内毒素引起，是致命的，40%的有症状的人死亡。认识到病原菌是致命的，容易扩散，并可用于生物恐怖主义，强调需要随时检测病原菌，并有解毒剂来预防它们。 抗体是检测特定内毒素分子的一种非常重要的手段。然而，抗体的产生和特异性是昂贵的，结果可能是可变的。有时，抗体也会在两个内毒素结构之间或与人体组织中的碳水化合物发生交叉反应，因此用途有限。在这里，我们建议开发识别特定内毒素结构的适体。适配子是核酸，像抗体一样，以高亲和力识别它们所针对的目标大分子。与抗体不同，适配子是在体外选择的，因此比抗体更容易被修饰，从而具有特定的所需特征。适配子还具有许多有利于诊断和现场工作的特征。我们对抗内毒素适配子的长期目标是，它们将为从细菌表面检测内毒素的能力增加一个新的维度，这些适配子也可能在被动免疫或其他医疗应用中使用。我们预计，作为未来公共场所现场检测系统的一部分，适体在均相分析和微阵列中的使用将用于病原体的现场检测，以早期检测生物恐怖活动。 这一应用的具体目的是：1)选择高特异性和高亲和力的适体，2)鉴定所选的适体，3)开发一种能检测特定内毒素寡糖的变构适配子钳。

DESCRIPTION (provided by applicant): Lipopolysaccharides are a distinguishing feature of gram negative bacterial surfaces. Antibodies that recognize and distinguish between different O-specific oligosaccharide units are used to identify pathogenic bacterial strains that are been found in contaminated foods and have caused human death and disease. LPS molecules are often intimately involved in pathogenesis and participate in functions such as bacterial adhesion and movement, and in eliciting autoimmune responses. Septic shock syndrome, a severe inflammatory response that is often elicited by LPS, is deadly, killing 40% of individuals with symptoms. Recognition that pathogenic bacteria are deadly, easily dispersed and could be used for bioterrorism emphasizes the need to readily detect pathogenic bacteria and of having antidotes to protect against them. Antibodies are a very important means of detecting specific LPS molecules. However, the production and specificity of antibodies is expense and results can be variable. On occasion, antibodies are also cross-reactive between two LPS structures or with carbohydrates in human tissues and thus have limited use. Here we propose to develop aptamers that recognize specific LPS structures. Aptamers are nucleic acids that, like antibodies, specifically recognize with high affinity the target macromolecules against which they were selected. Unlike antibodies, aptamers are selected ex-vivo and consequently can be more readily modified than antibodies so as to possess particular desired characteristics. Aptamers also possess many characteristics that are advantageous for diagnostics and field work. Our long term goal for anti-LPS aptamers is that they will add a new dimension to the ability to detect LPS from bacterial surfaces and that these aptamers also might find use in passive immunity or in other medical applications. We foresee the use of aptamers in homogeneous assays and in microarrays for on location detection of pathogens as a part of future in-place detection systems in public spaces for early detection of bioterrorist activities. The specific aims for this application are to: 1) Select for aptamers that bind LPS with high specificity and high affinity, 2) Characterize the selected aptamers, 3) Develop an allosteric aptamer CLAMP that detects a specific LPS oligosaccharide.