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

我们的主要目标是发展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。根据这一建议，我们的目标是通过在基本的苄基鸟嘌呤残基和磷酸二酯骨架上加入耐光保护基团来光控制RNA编辑(首先是在体外，后来在细胞培养中)。如果成功，这将使在光的空间和时间控制下，在细胞内组装具有酶功能的导向RNA-脱氨酶偶联物。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，在过去的两年里，我们已经建立了补骨脂素交联剂用于DNA功能的光控制。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，以便稍后将其引入更大的RNA结构。这可能允许我们用光来控制这种结构的平移。该方法有可能补充依赖于耐光性保护基团的经典方法，因此可能提供新的应用途径。与经典的光笼相比，已经有一个单一的交联键就可以可靠地、完全地将核酸冻结在一个能量上不受欢迎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.