Nano-Antibodies for Studying RNA Modifications

描述（由申请人提供）：拟议I期研究的目标是开发一套商用试剂，用于识别2'- o -甲基腺苷（Am）， 2'- o -甲基胞苷（Cm）， 2'- o -甲基鸟苷（Gm）或2'- o -甲基尿苷（Um），以及不加区分地识别所有4个2'- o -甲基核糖片段。作为这些新试剂基础的核心技术是基于骆驼抗体的独特特性，骆驼抗体已被广泛接受为蛋白质和半抗原的有价值的结合试剂，但到目前为止还没有任何RNA修饰的例子。所提出的来自camelidae的单域（VHH）抗体的主要优点是它们具有非常高的亲和力和特异性，并且它们易于作为单克隆抗体复制，稳定且体积很小，当缀合到染料中时可以实现超分辨率成像。目前，任何类型的亲和试剂都只能用于监测100多个RNA修饰片段中的一小部分。这些可用试剂的最佳例子是n6 -甲基腺苷（m6A）抗体，它被证明对该领域的研究人员具有重要价值

DESCRIPTION (provided by applicant): The goal of the proposed Phase I research is to develop a set of commercial reagents for recognizing 2'-O-methyladenosine (Am), 2'-O-methylcytidine (Cm), 2'-O-methylguanosine (Gm) or 2'-O-methyluridine (Um), as well as indiscriminately to all four 2'-O-methyl ribose moiety. The core technologies that serve as the foundation of these new reagents are based on the unique properties of camelid antibodies, which have been well accepted as valuable binding reagents for proteins and haptens, but so far without any example for RNA modifications. The main advantage of the proposed single-domain (VHH) antibodies from camelidae is that they have very high affinity and specificity, and they are easy to reproduce as monoclonal antibodies, stable, and of very small size, enabling super-resolution imaging when conjugated to dye. Currently, affinity reagents of any type are only available for monitoring a handful among the more than 100 RNA modification moieties. The best examples of such available reagents are the antibodies against N6-methyladenosine (m6A), which proved to be of significant value to researchers in the field trying to understand the functions of the m6A-modified mRNAs. However, the two anti-m6A antibodies, created about 3 decades ago, are both rabbit polyclonal antibodies that are not easily reproducible or suitable for high precision immune-imaging or other modern assays. There is an urgent need for development of high quality antibodies against the fast-expanding and increasingly important family of known modified mRNAs and regulatory RNAs. Successful execution of the proposed project will establish a fully tested, market-ready group of reagents and kits for detecting one type of closely related RNA modifications, 2'-O-methylation on the ribose of the 4 RNA bases. Currently, there is a lack of such reagents on the market, providing the applicant a very good chance of commercial success with the outcome of the proposed project. After achieving this goal, similar efforts can be initiated in a phase II SBIR or other types of project to develop more nano-antibodies against additional RNA modifications, eventually overcome the bottleneck of post-transcriptional RNA studies, and serve as the foundation for advancing NIDA- and NIMH-relevant research projects on RNA influence in various substance abuse or mental diseases.