Directed Evolution of an Epitranscriptomic Toolkit

项目摘要/摘要 RNA的转录后修饰对RNA的稳定性、转运和翻译为 蛋白质。在人类中，最普遍的RNA修饰N6-甲基腺苷(M6A)的破坏已经被 与癌症等疾病有关。为了更好地了解m6A在疾病发病机制中的作用，生物学家 需要强大的方法来定位RNA中的m6A位点。尽管最近取得了进展，但现有的方法需要 步骤繁多，缺乏选择性和敏感性，或表现出序列偏差。这个项目的长期目标是 是通过开发能够检测m6A的酶并将这些工具应用于 癌细胞的表位转录分析。两个战略将并行进行：1)反向发展- 转录酶，它以RNA为模板聚合DNA，将核苷酸“标记”合并到DNA中 在细胞RNA反转录过程中的m6A位点。这将有助于直接读出m6A，而不需要 对基于抗体的下拉的要求。2)设计脱氨酶标记m6A位点。腺嘌呤脱氨酶 将腺嘌呤(A)转化为肌苷(I)，聚合酶将其读作鸟嘌呤(G)。因此，工程脱氨酶 选择性地对M6A而不是A进行脱氨基将产生一个可以通过RNA测序定位的标记。 最后，进化的逆转录酶和脱氨酶平台将彻底生化 为哺乳动物细胞m6A测序的特征化和实施化建立了建议的平台。这个 开发和传播这些工具将通过促进对m6A及其作用的研究而产生广泛的影响 在人类健康方面。

PROJECT SUMMARY/ABSTRACT The post-transcriptional modification of RNA is critical for RNA stability, trafficking, and translation into proteins. In humans, disruption of the most prevalent RNA modification, N6-methyladenosine (m6A), has been linked to diseases such as cancer. To better understand the role of m6A in disease pathogenesis, biologists require robust methods for mapping m6A sites within RNA. Despite recent progress, existing methods require numerous steps, lack selectivity and sensitivity, or demonstrate sequence bias. The long-term goal of this project is to address these limitations by developing enzymes that can detect m6A and applying these tools for epitranscriptomic analysis of cancer cells. Two strategies will be pursued in parallel: 1) Evolve reverse- transcriptases, which polymerize DNA using RNA as a template, to incorporate a nucleotide “marker” into DNA at m6A sites during reverse transcription of cellular RNA. This would facilitate direct readout of m6A without the requirement for antibody-based pulldowns. 2) Engineer deaminases to mark m6A sites. Adenine deaminases convert adenine (A) to inosine (I), which is read as guanine (G) by polymerases. Thus, engineering deaminases to selectively deaminate m6A, rather than A, would result in a mark that can be located by RNA sequencing. Finally, the evolved reverse transcriptase and deaminase platforms will be thoroughly biochemically characterized and implemented for m6A sequencing of mammalian cells to establish the proposed platform. The development and dissemination of these tools will have a broad impact by facilitating studies of m6A and its role in human health.