Functional and structural characterization of aptamers and their applications in DNA assemblies

核酸是用途极其广泛的分子。DNA和RNA除了在信息存储和传递中发挥作用外，还作为功能分子发挥着积极的作用。它们可以作为催化剂(核酶，DNAzyme)，参与配体结合(适配子，核糖开关)，并作为构建纳米级分子器件的材料(DNA折纸)。然而，功能核酸通常被认为是一个黑匣子。这些类型的分子如何工作的细节通常是未知的。它们的结构，它们如何与其他分子相互作用，以及它们如何折叠成活性分子，人们往往知之甚少。我实验室的研究旨在解决这种缺乏理解的问题，并了解功能核酸分子如何执行其复杂的任务。作为一个发现功能核酸如何工作的模型系统，我们的工作重点是了解可卡因结合适体的工作原理。*可卡因结合DNA适体的结构由一个包含串联AG错配的三向连接组成。可卡因结合适体的一个不同寻常的特征是其结合的混杂。最初被选择与可卡因结合，这种适体结合奎宁的强度增加了50倍。这个适配子也很有趣，因为它的结合机制取决于它的一个茎的长度。当茎1为三个碱基对或更短时，它经历配体诱导的折叠。当它包含四个或更多碱基对时，该结构是预先形成的。本研究旨在全面定义当茎1的长度变化时适体结合和折叠的热力学。我们将研究适配子的自由形式、奎宁结合形式和可卡因结合形式，以确定结合的自由能垒如何随着结合机制的变化而变化。自由和配体结合的可卡因适体的三维结构将使用核磁共振和小角X射线散射(SAXS)技术相结合的方法来确定。这些结构不仅对适配子的结构提供了重要的见解，而且对它如何与不同的配体相互作用提供了重要的见解。我们还打算应用我们对该适配子结构和功能的了解，以受控和设计的方式组装一系列适配子。这项工作将对分子识别以及使用适配子作为生物传感器的实际应用产生基本的见解。我的研究计划的一个长期目标是将这些研究扩展到其他适体-配体系统，以便制定不同功能核酸分子如何工作的一般规则。

Nucleic acids are tremendously versatile molecules. Aside from their role in information storage and transfer, DNA and RNA play active roles as functional molecules. They can act as catalysts (ribozymes, DNAzymes), participate in ligand binding (aptamers, riboswitches), and play a role as material for building nanoscale molecular devices (DNA origami). However, functional nucleic acids are typically thought of as a black box. The fine details of how these types of molecules work are often unknown. Their structures, how they interact with other molecules and how they fold into an active molecule are often poorly understood. Research in my laboratory aims to tackle this lack of understanding and learn how functional nucleic acid molecules perform their complex tasks. As a model system for discovering how functional nucleic acids work, our work is focusing on understanding how the cocaine-binding aptamer operates.***The structure of the cocaine biding DNA aptamer is composed of a three-way junction containing a tandem AG mismatch. An unusual feature of the cocaine-binding aptamer is its binding promiscuity. Originally selected to bind cocaine, this aptamer binds quinine 50-fold tighter. This aptamer is also interesting in that its binding mechanism depends on the length of one of its stems. When stem 1 is three base pairs or shorter, it undergoes ligand-induced folding. When it contains four or more base pairs, the structure is pre-formed. The research proposed here aims to fully define the thermodynamics of binding and folding of the aptamer as the length of stem 1 is varied. We will study the free, quinine-bound and cocaine-bound forms of the aptamer in order to determine how the free energy barrier to binding changes as the binding mechanism changes. The three-dimensional structure of the free and ligand-bound cocaine-binding aptamer will be determined using a combination of nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS) techniques. These structures will provide important insights not only into the structure of the aptamer but in how it interacts with different ligands. We also aim to apply our knowledge of the structure and function of this aptamer to assemble a chain of aptamers in a controlled and designed manner. This work will produce fundamental insights in to molecular recognition as well as practical applications in using aptamers as biosensors. A long-term aim of my research program is to expand these studies to other aptamer-ligand systems in order to formulate general rules for how different functional nucleic acid molecules work.