Unnatural Base Pairs as DNA Bioprobes

描述(由申请人提供)： 这一提议旨在利用一系列非天然核苷来询问核酸的结构和功能。从纳尔逊·伦纳德的苯基腺苷类似物中获得灵感，我们将通过在天然的嘌呤和嘧啶支架中引入多样性来改变核苷酸碱基的宽度，并随后改变DNA螺旋的宽度。将吡咯、噻吩环或呋喃间隔环引入到杂芳烃结构中将提供以前研究的碱基对所不能提供的独特的结构优势。这项研究将通过对DNA螺旋稳定性的物理方面以及静电和碱基堆积所起的作用提供新的见解来解决一些基本的科学问题。它还将提供关于碱基形状、碱基堆积和静电对DNA聚合酶复制的影响的有用数据。我们预测，由于静电表面积的增加，以及色散和范德华力和极化率的增加，杂芳环提供的优势将大大有助于增强碱基堆积。此外，杂芳族间隔环具有与阳离子或存在于螺旋水化骨架中的水形成氢键的能力。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，可能在许多应用中被证明是有用的，例如螺旋形成的自报告程序，跟踪单点突变，以及在合成DNA微阵列中。这项研究的最初目标是：(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)将非天然的核苷引入不同长度和组成的DNA双链中；(Iii)从计算、光谱、热力学和结构上表征所得到的螺旋；以及(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.