In Vitro Evolution of Functional Biomolecules Using CE

描述（由申请人提供）：SELEX是一种组合化学的进化方法，它使用体外选择来鉴定对特定靶标具有亲和力的RNA或DNA序列。这些功能序列，也被称为适体，已经被用作作用于特定生物受体的药物，或作为可用于生物医学分析或成像的诊断试剂。本文首次提出了一种在自由溶液中选择功能DNA分子的独特方法（CE-SELEX）。选择将根据活性DNA序列和目标之间的相互作用引起的电泳迁移率转移进行，在自由溶液中进行选择将消除传统SELEX中色谱选择引入的许多进化偏差。色谱分离带来的最明显的偏差是选择不是针对实际目标进行的。相反，选择对附着在固定支架上的目标具有亲和性的序列。另一个问题是动力学偏差，其中几乎不可能从色谱柱中洗脱非常强的相互作用序列。自由溶液中的电泳选择将消除这些偏差，提供具有改进的结合效率和选择性的适体。CE-SELEX提高的灵活性也将使选择过程的基本原理首次得到深入研究，进一步提高所选适配体的质量。首先，将使用20碱基的ssDNA作为靶标进行对照实验，以确保CE-SELEX选择最佳结合序列。使用CE-SELEX选择的适配体的亲和力将与使用常规SELEX获得的适配体进行比较，使用大（lgE）和小（ATP）靶标。使用CE-SELEX选择的适配体的选择性将通过搜索特异性结合n -甲基间卟啉而不是中卟啉，d -丝氨酸而不是l -丝氨酸以及g-ABA而不是a- aba和b-ABA的序列来测试。最后，将优化影响最终适配体结合效率和选择性的选择条件，包括靶浓度、初始DNA池大小和负选择。预计通过消除固定相在常规SELEX中引入的偏差，CE-SELEX将提供具有更高结合效率和选择性的适配体。提高使用SELEX筛选的适体的质量将对健康研究有直接的好处。提高结合效率和选择性将有利于开发作用于特定生物受体的适体药物。具有更好的结合效率和选择性的适体可能显示出更高的药理活性和更少的副作用。改进的适体也将在许多领域被用作诊断标记，包括医学分析、体内成像和生物传感器。

DESCRIPTION (provided by applicant): SELEX is an evolutionary approach to combinatorial chemistry that uses in vitro selection to identify RNA or DNA sequences with affinity for a particular target. These functional sequences, also referred to as aptamers, have found use as drugs that act on specific biological receptors or as diagnostic agents that can be used in biomedical analyses or imaging.This proposal outlines a unique method (CE-SELEX) for selecting functional DNA molecules in free solution for the first time. Selection will be made on the basis of an electrophoretic mobility shift induced by interactions between active DNA sequences and the target performing the selection in free solution will eliminate many of the evolutionary biases introduced by the chromatographic selection in conventional SELEX. The most obvious bias introduced by chromatographic separation is that selection is not performed against the actual target. Instead a sequence is selected to have affinity for the target attached to a stationary support. Another concern is kinetic bias where it is almost impossible to elute very strongly interacting sequences from a chromatography column. Electrophoretic selection in free solution will eliminate these biases, providing aptamers with improved binding efficiency and selectivity. The improved flexibility of CE-SELEX will also allow the fundamentals of the selection process to be studied intensively for the first time, further improving the quality of the selected aptamers.Initially a control experiment using a 20-base ssDNA as a target will be performed to ensure that CE-SELEX does select for the optimum binding sequence. The affinities of aptamers selected using CE-SELEX will then be compared to those obtained using conventional SELEX using both a large (lgE) and small (ATP) target The selectivity of aptamers selected using CE-SELEX will be tested by searching for sequences that specifically bind N-methylmesoporphyrin over mesoporphyrin, D-serine over L-serine and g-ABA over a-ABA and b-ABA. Lastly, selection conditions expected to affect the binding efficiency and selectivity of the resulting aptamers, including target concentration, size of the initial DNA pool and negative selections, will be optimized. It is anticipated that by removing biases introduced by the stationary phase in conventional SELEX, CE-SELEX will provide aptamers with improved binding efficiency and selectivity.Improving the quality of aptamers selected using SELEX will have direct benefits to health research. Increased binding efficiency and selectivity will be beneficial in developing aptamer drugs that act on specific biological receptors. Aptamers with improved binding efficiency and selectivity may show increased pharmacological activity with fewer side effects. Improved aptamers will also find use in many areas as diagnostic markers including medical analyses, in vivo imaging and biosensors.