Extended Thermodynamic Database for Modified Oligonucleotides

描述（由申请人提供）：本项目将开发一个极大扩展的修饰寡核苷酸热力学数据库，并将其纳入现有的设计软件中。该快速通道SBIR提案侧重于确定目前广泛应用的修改的热力学参数。新的热力学数据库和软件将使世界各地的大量研究人员能够更容易地开发具有修饰寡核苷酸的先进应用程序，并将为软件产品增加大量的商业价值。在这个新的提案中，修饰寡核苷酸的范围将大大增加，包括各种非核苷酸连接物和13个已经发现最广泛应用的修饰核苷酸。这些修饰包括改善热力学稳定性的修饰核苷酸（2,6-二氨基嘌呤，2'- o -甲基RNA， 5-丙基嘧啶），通用碱（5-硝基吲哚和3-硝基吡啶），用于治疗的修饰寡核苷酸（磷硫酸酯，2'- o -甲基RNA， 2'-氟嘧啶）和用于其他用途的修饰寡核苷酸（2-氨基嘌呤，2-硫胸腺嘧啶，5-甲基胞嘧啶，7-去氮鸟嘌呤）。这项工作补充了在之前的项目（编号为R44GM076745）中测量了热力学参数的其他修饰核苷酸的工作。这项研究包括对500多个经过改良的双酶体以及未经过改良的对照组进行系统的一系列测量。新确定的参数将被纳入软件数据库，以便在比以前可能的更广泛的修饰寡核苷酸覆盖范围下进行杂交实验的模拟和计算机优化。这将有助于研究人员设计更可靠的检测和治疗方法。修饰的寡核苷酸可以根据修饰的核苷酸的结构或其预期的应用进行分类。在这项拟议的研究中，通过结构和功能检查了最常用的修饰的代表性样本。虽然许多修饰相互结合使用，但不建议在本项目中研究同一寡核苷酸内不同修饰的组合，因为可能的组合数量将使这项任务无法成立。在组合中使用的修饰的一些例子包括：2'- o -甲基- rna含有末端硫代磷酸，LNA-2'- o -甲基- rna组合，和硫代磷酸- rna组合。除了测量以前未测量的寡核苷酸的热力学参数外，还需要重新构建软件建模和模拟程序，以允许在程序的计算和显示模块中包含这些核苷酸。公共卫生相关性：本项目旨在确定天然RNA与与DNA或RNA相关的修饰合成化合物之间的热力学结合能，以促进各种生化分析。这些数据将被纳入我们现有的软件设计和建模的寡核苷酸分析和杂交。该软件的增强将使该软件在设计反义治疗、RNAi敲除、法医学和许多其他应用的检测时更有用。

DESCRIPTION (provided by applicant): A greatly expanded thermodynamic database for modified oligonucleotides will be developed in this project and incorporated into existing design software. This Fast Track SBIR proposal focuses on determining thermodynamic parameters for modifications that are currently finding widespread applications. The new thermodynamic database and software will enable a large number of researchers throughout the world to more easily develop advanced applications with modified oligonucleotides and will add substantial commercial value to the software products. In this new proposal, the range of modified oligonucleotides will be greatly increased to include a variety of non-nucleotide linkers and 13 additional modified nucleotides that have found the widest applications. These modifications include modified nucleotides that improve thermodynamic stability (2,6- diaminopurine, 2'-O-methyl RNA, 5-propynylpyrimidines), universal bases (5-nitroindole and 3-nitropyrole), modified oligonucleotides used in therapeutics (phosphorothioates, 2'-O-methyl RNA, 2'-fluoropyrimidines) and modified oligonucleotides with other applications (2-aminopurine, 2-thiothymine, 5-methylcytosine, 7- deazaguanine). This work complements the work done on other modified nucleotides whose thermodynamic parameters were measured during a previous project (under R44GM076745). The study involves a systematic series of measurements on over 500 modified duplexes along with unmodified controls. The newly determined parameters will be incorporated into a software database to enable simulation and in silico optimization of hybridization experiments in the presence of a wider coverage of modified oligonucleotides than previously possible. This will aid researchers in designing more robust assays and therapeutics. Modified oligonucleotides can be categorized either by the structure of the modified nucleotide or by its intended application. In this proposed research, a representative sample of the most commonly used modifications, both by structure and by function, is examined. Although many modifications are used in combination with one another, it is not proposed to study combinations of different modifications within the same oligonucleotide in this project as the number of possible combinations would make this task untenable. Some examples of modifications used in combination include: 2'-O-methyl-RNA containing terminal phosphorothioate, LNA-2'-O-methyl-RNA combinations, and phosphorothioate-LNA combinations. In addition to the measurements of thermodynamic parameters for oligonucleotides not previously measured, it will be necessary to restructure the software modeling and simulation program to allow for the inclusion of these nucleotides in both calculation and display modules of the program. PUBLIC HEALTH RELEVANCE: This project is designed to determine the thermodynamic binding energies between natural RNA and modified synthetic compounds related to DNA or RNA designed to facilitate various biochemical assays. This data will then be incorporated into our existing software for design and modeling of oligonucleotide assays and hybridization. This enhancement to the software will make the software more useful in designing assays for antisense therapeutics, RNAi knockdown, forensics, and many other applications.