In Vitro Evolution of Functional Biomolecules Using CE

使用 CE 进行功能生物分子的体外进化

• 批准号: 6604936
• 负责人: MICHAEL T BOWSER
• 金额: $ 24.97万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6604936
• 关键词: DNA binding protein; RNA binding protein; adenosine triphosphate; aminobutyrate; capillary electrophoresis; cell line; combinatorial chemistry; directed evolution; gel mobility shift assay; genetic techniques; immunoglobulin E; ligands; method development; nucleic acid sequence; oligonucleotides; polymerase chain reaction; serine

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.

描述（由申请人提供）：SELEX是组合化学的一种进化方法，其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，已发现用作药物，作用于特定的生物受体或作为诊断剂，可用于生物医学分析或imaging.This proposal概述了一个独特的方法（CE-SELEX）选择功能的DNA分子在自由溶液中的第一次。选择将基于由活性DNA序列之间的相互作用诱导的电泳迁移率变化进行，并且在游离溶液中进行选择的靶将消除由常规SELEX中的色谱选择引入的许多进化偏差。色谱分离引入的最明显的偏倚是选择不是针对实际目标进行的。相反，选择对附着于固定支持物的靶具有亲和力的序列。另一个问题是动力学偏差，其中几乎不可能从色谱柱中分离出非常强的相互作用序列。在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的灵活性的提高也将使选择过程的基本原理首次得到深入研究，进一步提高所选适体的质量。最初将使用20个碱基的ssDNA作为靶进行对照实验，以确保CE-SELEX确实选择最佳结合序列。然后将使用CE-SELEX选择的适体的亲和力与使用常规SELEX使用大（IgE）和小（ATP）靶标获得的亲和力进行比较。使用CE-SELEX选择的适体的选择性将通过搜索特异性结合N-甲基中卟啉超过中卟啉、D-丝氨酸超过L-丝氨酸和g-ABA超过a-ABA和b-ABA的序列来测试。最后，将优化预期影响所得适体的结合效率和选择性的选择条件，包括靶浓度、初始DNA池的大小和阴性选择。CE-SELEX通过消除传统SELEX中固定相引入的偏差，有望提供具有更高结合效率和选择性的适体，提高SELEX筛选适体的质量将对健康研究产生直接的效益。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。具有改善的结合效率和选择性的适体可以显示出增加的药理学活性和较少的副作用。改进的适体还将在许多领域中用作诊断标记物，包括医学分析、体内成像和生物传感器。