Unnatural Base Pairs as DNA Bioprobes

描述（由申请人提供）： 该方案旨在利用一系列非天然核苷来研究核酸的结构和功能。从纳尔逊伦纳德的苯扩展腺苷类似物的灵感，我们将改变核碱基的宽度，随后的DNA螺旋，通过引入多样性到天然嘌呤和嘧啶支架。将吡咯、噻吩或呋喃间隔环并入杂芳族结构中将提供先前研究的碱基对不可能具有的独特结构优势。这项研究将通过提供对DNA螺旋稳定性的物理方面以及静电和碱基堆积所起作用的新见解来解决一些基本的科学问题。它也将提供有用的数据，核碱基的形状，碱基堆积和静电对DNA聚合酶复制的影响。我们预测，由杂芳族间隔环提供的优点将有助于显着增强的基础堆叠，由于增加的静电表面积，以及增加的分散和货车范德华力和极化率。此外，杂芳族间隔环具有与阳离子或与存在于螺旋水合的脊中的水氢键合的能力。因此，我们还预测将观察到DNA螺旋稳定性的显著增加。此外，这些延伸的碱基是荧光的，可以证明在许多应用中是有用的，例如螺旋形成的自我报告，跟踪单点突变，以及合成DNA微阵列。本研究的最初目的是（i）设计，合成和表征一系列扩展的嘧啶和扩展的嘌呤核苷;（ii）将非天然核苷掺入不同长度和组成的DNA双链体中;（iii）计算，光谱，化学和结构表征所得的螺旋;和（iv）评估它们对DNA聚合酶保真度的影响。

DESCRIPTION (provided by applicant): This proposal is aimed at using a series of unnatural nucleosides to interrogate nucleic acid structure and function. Drawing inspiration from Nelson Leonard's benzene-expanded adenosine analogues, we will vary the width of the nucleobases, and subsequently the DNA helix, by introduction of diversity into the natural purine and pyrimidine scaffolds. Incorporation of either a pyrrole, thiophene or furan spacer ring into the heteroaromatic architecture will provide unique structural advantages not possible with previously studied base pairs. This research will address some fundamental scientific questions by providing new insights into the physical aspects of DNA helix stability and the role that electrostatics and base stacking play. It will also provide useful data regarding the effect nucleobase shape, base stacking and electrostatics have on DNA polymerase replication. We predict that the advantages provided by the heteroaromatic spacer rings will contribute significantly to enhanced base stacking, due to increased electrostatic surface area, as well as increased dispersion and van der Waals forces and polarizability. Furthermore, the heteroaromatic spacer rings possess the ability to hydrogen bond with cations or with water present in the spine of hydration of the helix. As a result, we also predict a significant increase in DNA helix stability will be observed. In addition, these extended bases are fluorescent and could prove useful in many applications, such self-reporters of helix formation, tracking single point mutations, as well as in synthetic DNA microarrays. The initial aims of this study are to (i) design, synthesize and characterize a series of extended pyrimidines and expanded purine nucleosides; (ii) to incorporate the unnatural nucleosides into DNA duplexes of varying length and composition; (iii) to computationally, spectroscopically, thermodynamically and structurally characterize the resulting helices; and (iv) evaluate their effect on DNA polymerase fidelity.