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: 6915670
• 负责人: MICHAEL T BOWSER
• 金额: $ 24.94万
• 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/6915670
• 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(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与特定目标具有亲和力的RNA或DNA序列。这些功能序列也被称为适配子，已被用作作用于特定生物受体的药物或可用于生物医学分析或成像的诊断试剂。这项建议概述了一种独特的方法(CE-SELEX)，首次在自由溶液中选择功能DNA分子。选择将根据活性DNA序列之间的相互作用引起的凝胶迁移率变化进行，而在自由溶液中执行选择的目标将消除传统SELEX中由层析选择引入的许多进化偏见。层析分离带来的最明显的偏差是不针对实际目标进行选择。取而代之的是，选择与固定载体上连接的靶具有亲和力的序列。另一个令人担忧的问题是动力学偏差，在这种情况下，几乎不可能从色谱柱上洗脱出相互作用非常强的序列。在无水溶液中的电泳法选择将消除这些偏向，为适体提供更高的结合效率和选择性。CE-SELEX灵活性的提高也将使人们能够第一次深入研究选择过程的基本原理，进一步提高所选适配子的质量。首先，将进行以20个碱基的单链DNA为靶标的对照实验，以确保CE-SELEX确实选择了最佳结合序列。然后，使用CE-SELEX选择的适配子的亲和力将与使用大(LGE)和小(ATP)靶标的常规SELEX获得的适配子的亲和力进行比较。使用CE-SELEX选择的适配子的选择性将通过搜索N-甲基中卟啉与中卟啉、D-丝氨酸与L-丝氨酸以及g-脱落与a-脱落酸和b-脱落酸结合的序列来测试。最后，将优化影响所得适配子结合效率和选择性的选择条件，包括目标浓度、初始DNA池的大小和负选择。通过消除传统SELEX中固定相引入的偏差，CE-SELEX将为适配子提供更高的结合效率和选择性，改善使用SELEX选择的适配子的质量将对健康研究有直接好处。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。具有更高的结合效率和选择性的适配子可能会显示出更高的药理活性和更少的副作用。改进的适体还将在许多领域用作诊断标记，包括医学分析、活体成像和生物传感器。