Pseudo-Complementary DNA

描述(申请人提供)：从概念上讲，伪互补DNA(PcDNA)是一种由碱基类似物组成的“无结构”聚合物，这些碱基类似物彼此不相互作用，但能够在互补探针中将Watson-Crick配对到规则碱基。得到的杂交物中的碱基对由修饰核苷酸和天然核苷酸的独特组合组成。将天然DNA或RNA转化为无结构的pcDNA的能力将显著提高短探针的可靠性和效率，因为它消除了靶中二级结构的干扰。这种探针区分错配的精妙能力有利于它们在单核苷酸多态的直接检测中的使用。PcDNA靶标还将促进通用寡核苷酸微阵列的实际使用和极高密度SNP微阵列的开发。基于寡核苷酸连接或通过纳米孔的高通量测序技术也将受益。最近，我们开发了一系列dNTP类似物，支持DNA的酶合成，减少了二级结构，增加了对短的未经修饰的探针的可及性。在这项拨款申请中，我们建议优化pcDNA的制备方法，并从可获得性、稳定性和特异性方面严格表征这种DNA的杂交特性。将花费大量的努力来开发一种新的、快速且经济有效的方法来确定新碱基的配对和错配的最近邻自由能，例如用于制备pcDNA的碱基。这种方法将使用微阵列平台来同时确定数百个短双链的熔融分布。这项研究计划的成功完成将提供对伪互补核酸的实用性和益处的全面分析，并确定微阵列是否可以用于简化和加速杂交自由能参数的获取。 公共卫生相关性：被称为寡核苷酸的短核酸片段因其与DNA和RNA相互作用的能力而在人类诊断、药物遗传学和高通量测序中得到应用。不幸的是，由于自然产生的DNA和RNA中存在高阶结构，寡核苷酸的性能受到了影响。在这笔赠款中，我们将评估一种消除DNA高阶结构的新策略，从而提高寡核苷酸在新的和现有的应用中的效用。在将这项技术付诸实践的过程中，我们将测试一种基于微阵列的新策略，以获取描述寡核苷酸与“无结构”DNA相互作用的热力学参数。这个热力学图书馆将促进这项新技术的使用。

DESCRIPTION (provided by applicant): Conceptually, pseudo-complementary DNA (pcDNA) is a "structure-free" polymer composed of base analogs that don't interact with each other but are able to Watson-Crick pair to regular bases in a complementary probe. Base pairs in the resulting hybrids are composed of unique combinations of modified and natural nucleotides. The ability to convert native DNA or RNA into structure-free pcDNA would significantly improve the reliability and efficiency of short probes by eliminating interference from secondary structure in the target. The exquisite ability of such probes to discriminate against mismatches favors their use in the direct detection of single nucleotide polymorphisms. pcDNA targets would also facilitate the practical use of universal oligonucleotide microarrays and the development of extremely dense SNP microarrays. High throughput sequencing technologies based on oligonucleotide ligation or passage through a nanopore would also benefit. Recently, we developed a set of dNTP analogs that support enzymatic synthesis of DNA with reduced secondary structure and increased accessibility to short unmodified probes. In this grant application we propose to optimize methods for the preparation of pcDNA and to rigorously characterize the hybridization properties of this DNA with respect to accessibility, stability and specificity. Significant effort will be expended in developing a new, rapid and cost-effective approach to determining nearest neighbor free energies for both pairing and mis-pairing of novel bases such as the ones used to prepare pcDNA. This approach will use a microarray platform to simultaneously determine the melting profiles of hundreds of short duplexes. Successful completion of this research program will provide a comprehensive analysis of the practicality and benefits of pseudo-complemementary nucleic acids and establish whether microarrays can be used to simplify and accelerate the acquisition of free energy parameters for hybridization. PUBLIC HEALTH RELEVANCE: Short pieces of nucleic acid known as oligonucleotides have applications in human diagnostics, pharmacogenetics and high throughput sequencing due to their ability to interact with DNA and RNA. Unfortunately, the performance of oligonucleotides is impaired by the presence of higher order structure in naturally occurring DNA and RNA. In this grant we will evaluate a novel strategy for eliminating higher order structure in DNA thereby improving the utility of oligonucleotides for both new and existing applications. In reducing the technology to practice, we will test a new microarray based strategy for acquiring the thermodynamic parameters that describe the interaction of oligonucleotides with "structure-free" DNA. This thermodynamic library will facilitate use of the new technology.