Posttranscriptional Control of Gene Function With Light-dependent RNA Editing and Reversal of Psoralen Crosslinks

我们的主要目标是开发利用光控制转录后基因调控的方法。最近，我们已经建立了定向A-to-I-RNA编辑作为转录修复和RNA重编程的工具。有了这个建议，我们的目标是光控RNA编辑（首先在体外，后来在细胞培养），通过将光不稳定的保护基团的基本苄基鸟嘌呤残基和磷酸二酯骨干。如果成功的话，这将使得在光的空间和时间控制下，在细胞内组装酶功能性向导RNA-脱氨酶缀合物成为可能。这将为许多有吸引力的应用铺平道路，甚至超越RNA编辑。此外，在过去的两年中，我们已经建立了用于光控制DNA功能的Pupen-crosslinking。我们现在的目标是将这项技术转移到RNA水平。为此，我们将产生一个小的交联发夹基序，用于随后将其引入更大的RNA构建体。这可能使我们能够控制这种结构与光的平移。该方法有可能补充经典的方法，依赖于光不稳定的保护基团，因此可能提供新的应用。与经典的光笼化相反，单个交联已经可以可靠地且完全地将核酸冻结在能量上不利的二级和三级结构中。

Our major objective is the development of methods for controlling posttranscriptional gene regulation with light. Recently, we have established directed A-to-I-RNA editing as a tool for transcript repair and RNA reprogramming. With this proposal we aim to photocontrol RNA editing (first in vitro, later in cell culture) by incorporating photolabile protection groups on the essential benzylguanine residue and on the phosphordiester backbone. If successful this will enable the assembly of enzymatically functional guideRNA-deaminase conjugates inside the cell under spatial and temporal control with light. This would pave the way towards numerous attractive applications even beyond RNA editing.Furthermore we have established Psoralen-crosslinking for the photocontrol of DNA function during the last two years. We now aim to transfer the technology to the RNA-level. For this we will generate a small crosslinked hairpin motif for its later introduction into larger RNA constructs. This may allow us to control the translation of such constructs with light. The method has the potential to complement classical methods that rely on photolabile protection groups and may thus offer access to novel applications. In contrast to classical photocaging, already a single crosslink can freeze a nucleic acid reliably and completely in an energetically unfavored secondary and tertiary structure.