Development of cap-caged-mRNA for optochemical control of translation in eukaryotic cells

光是一种多功能的调节元件，因为它与大多数细胞成分完全正交，非侵入性，在组织、细胞甚至亚细胞区域的时间和定位上是可控的。不同的光化学方法已经可以通过时空控制在DNA、mRNA和蛋白质水平上控制基因表达。然而，目前还没有一种有效的时空调控的方法来诱导外源mRNA的翻译。我们提出了一种创新的、高度灵活的方法来触发时空控制的外源mRNA的翻译。为此，我们将开发一种化学-酶方法来用光切割基团修饰mRNA帽，并完全阻断mRNAs的翻译。我们将使用高度杂乱的N7帽甲基转移酶ECM1，并合成新的共底物S-腺苷蛋氨酸类似物(ADOMet)。根据我们之前的工作，我们知道帽的N7位的修饰可以完全阻止翻译，因为它们取消了帽和翻译起始因子eIF4E之间的微妙相互作用。然后，我们将建立光触发移除这些笼群，以重建规范的帽子，从而触发在哺乳动物细胞中相应的mRNA的有效翻译。我们将首先在体外建立我们的方法，然后使用可量化的报告基因-mRNAs在哺乳动物细胞中实施它。最后，我们想要找出我们的方法是否适合通过激活caspase-mRNAs的翻译来触发具有时空控制的生理过程(即细胞凋亡)。

Light is a versatile regulatory element because it is fully orthogonal to most cellular components, noninvasive, controllable in timing and localization to tissues, cells and even subcellular regions. Various optochemical approaches have allowed the control of gene expression at the level of DNA, mRNA, and protein with spatio-temporal control. However, an efficient method to induce translation of exogenous mRNA with spatio-temporal control is missing. We propose an innovative and highly flexible approach for triggering translation of exogenous mRNA with spatio-temporal control. Towards this end, we will develop a chemo-enzymatic route to modify the mRNA cap with photo-cleavable groups and completely block translation of mRNA. We will use the highly promiscuous N7 cap methyltransferase Ecm1 and synthesize novel analogs of the cosubstrate S-adenosyl methionine (AdoMet). Based on our previous work we know that modifications at the N7 position of the cap can completely block translation because they abrogate the delicate interaction between the cap and the translation initiation factor eIF4E. We will then establish light-triggered removal of these caging groups to reconstitute the canonical cap and thus trigger efficient translation of the respective mRNA in mammalian cells.We will first establish our approach in vitro and then implement it in mammalian cells using quantifiable reporter-mRNAs. Finally, we want to find out whether our approach will be suitable to trigger a physiological process (namely apoptosis) with spatio-temporal control, by activating translation of caspase-mRNAs.