Extended Thermodynamic Database for Modified Oligonucleotides

描述(由申请人提供)：该项目将开发一个大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。这项快速跟踪SBIR建议的重点是为目前正在得到广泛应用的修改确定热力学参数。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实质性的商业价值。在这项新提案中，修饰寡核苷酸的范围将大大增加，包括各种非核苷酸连接体和另外13个已发现最广泛应用的修饰核苷酸。这些修饰包括提高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)、通用碱基(5-硝基吲哚和3-硝基吡咯)、用于治疗的修饰寡核苷酸(硫代硫酸盐、2’-O-甲基RNA、2‘-氟嘧啶)以及具有其他用途的修饰寡核苷酸(2-氨基嘌呤、2-硫代胸腺嘧啶、5-甲基胞嘧啶、7-去氮鸟嘌呤)。这项工作是对上一个项目(根据R44GM076745)测量热力学参数的其他修饰核苷酸所做工作的补充。这项研究包括对500多个修改过的双链以及未修改的对照进行系统的一系列测量。新确定的参数将被合并到软件数据库中，以便在存在比以前可能的更广泛的修饰寡核苷酸的情况下，对杂交实验进行模拟和电子优化。这将有助于研究人员设计更可靠的分析和治疗方法。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用途进行分类。在这项拟议的研究中，考察了最常用修饰语的一个代表性样本，包括结构和功能。虽然许多修饰是相互结合使用的，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，因为可能组合的数量会使这项任务无法完成。组合中使用的一些修饰的例子包括：含有末端硫代磷的2‘-O-甲基-RNA、LNA-2’-O-甲基-RNA组合和硫代硫酸盐-LNA组合。除了测量以前没有测量过的寡核苷酸的热力学参数外，还需要重组软件建模和模拟程序，以便将这些核苷酸包括在程序的计算和显示模块中。

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.