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

光是一种通用的调节元件，因为它与大多数细胞组分完全正交，无创，在组织，细胞甚至亚细胞区域的定时和定位方面可控。各种光化学方法已经允许在DNA、mRNA和蛋白质水平上通过时空控制来控制基因表达。然而，缺乏一种有效的方法来诱导外源mRNA的翻译与时空控制。我们提出了一种创新的和高度灵活的方法来触发翻译的外源mRNA的时空控制。为此，我们将开发一种化学-酶促途径，用光可切割基团修饰mRNA帽，并完全阻断mRNA的翻译。我们将使用高度混杂的N7帽甲基转移酶Ecm 1和合成新的类似物的辅助底物S-腺苷甲硫氨酸（NAMET）。基于我们先前的工作，我们知道帽的N7位置处的修饰可以完全阻断翻译，因为它们废除帽和翻译起始因子eIF 4 E之间的微妙相互作用。然后我们将建立光触发去除这些笼状基团，以重建规范帽，从而触发哺乳动物细胞中相应mRNA的有效翻译。我们将首先在体外建立我们的方法，然后使用可定量的mRNAs在哺乳动物细胞中实施。最后，我们想知道我们的方法是否适合通过激活半胱天冬酶mRNA的翻译来触发具有时空控制的生理过程（即凋亡）。

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.