SBIR Phase I: Synthesis of Long RNA Oligonucleotides via Enzymatic Ligation

这个小型企业创新研究（SBIR）第一阶段项目旨在开发制备60-200碱基RNA/DNA寡核苷酸的方法。目前用于制备长寡核苷酸的方法受到阻碍其在生物科学中应用的限制。研究人员表示，迫切需要更长的材料纳入非自然的修改和非规范的替代。获得这些寡核苷酸对于分子生物学和基于核酸的疗法的持续发现至关重要。尽管有这样的需求，最可靠的合成方法，2-ACE化学，可以提供质量的寡核苷酸不超过50个碱基。该SBIR第一阶段项目将使用2-ACE RNA开发一种新的生物化学技术，使用RNA连接酶将寡核苷酸酶促拼接在一起。RNA底物将作为复合物的一部分与互补夹板偶联。该项目将评估连接位点的不同构象、酶对不同底物序列的耐受性、夹板的最佳设计和组成以及底物和辅因子的理想浓度。一旦在迭代过程中确定，最佳条件将被应用于合作者确定的更具挑战性的研究问题。这项研究将直接解决国家生物技术研究界资源严重不足的问题，预计该项目的商业应用将是多种多样和立即的。例如，长度为50-200个碱基的位点特异性修饰的RNA应该是研究人员的高需求，他们正在开发RNA靶向药物或其他RNA依赖性技术，这些技术超出了目前的合成手段。

This Small Business Innovation Research (SBIR) Phase I project seeks to develop methods to prepare 60-200 base RNA/DNA oligonucleotides. Current methods for preparing long oligonucleotides suffer from limitations that hinder their application in biological science. Investigators have expressed an immediate need for longer material incorporating unnatural modifications and non-canonical substitutions. Access to these oligonucleotides is critical for continued discoveries in molecular biology and nucleic acid based therapeutics. Despite such demand, the most reliable synthetic method, 2-ACE chemistry, can provide quality oligonucleotides no longer than 50 bases. This SBIR Phase I project will use 2-ACE RNA to develop a novel biochemical technique using RNA ligase to enzymatically splice oligonucleotides together. RNA substrates will be coupled as part of a complex with a complementary splint. This project will evaluate different conformations of the ligation site, the tolerance of the enzyme for different substrate sequences, the optimal design and composition of the splint, and the ideal concentrations of substrates and cofactors. Once determined in an iterative process, the best conditions will be applied to more challenging research problems identified by collaborators. This research will directly address a critical deficit in the resources available to the national biotechnology research community.The commercial applications of this project are expected to be varied and immediate. As example, site-specifically modified RNAs of 50-200 bases in length should be in high demand by investigators who are developing RNA-targeted drugs or other RNA-dependent technologies beyond the reach of current synthetic means.