Directed Evolution of an Epitranscriptomic Toolkit

项目总结/摘要 RNA的转录后修饰对于RNA的稳定性、运输和翻译成RNA是至关重要的。 proteins.在人类中，破坏最普遍的RNA修饰，N6-甲基腺苷（m6 A）， 与癌症等疾病有关。为了更好地了解m6 A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内的m6 A位点。尽管最近取得了进展，但现有方法需要 步骤多，缺乏选择性和灵敏度，或显示序列偏差。这个项目的长期目标是 是通过开发可以检测m6 A的酶来解决这些限制，并将这些工具应用于 癌细胞的表观转录组分析。两个战略将并行实施：1）逆向发展- 一种转录酶，它以RNA为模板复制DNA，将核苷酸“标记”掺入DNA中 在细胞RNA逆转录过程中的m6 A位点。这将有助于直接读出m6 A，而不需要 需要基于抗体的下拉。2)工程脱氨酶以标记m6 A位点。腺嘌呤脱氨酶类 将腺嘌呤（A）转化为肌苷（I），通过聚合酶将其读作鸟嘌呤（G）。因此，工程化脱氨酶 选择性地将m6 A而不是A脱氨基，将产生可以通过RNA测序定位的标记。 最后，进化的逆转录酶和脱氨酶平台将被彻底地生物化学地 表征并实施用于哺乳动物细胞的m6 A测序以建立所提出的平台。的 这些工具的开发和推广将通过促进对m6 A及其作用的研究而产生广泛的影响 对人类健康的影响。

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.