Genetic code expansion for protein-protein and protein-DNA interaction studies in dynamic cellular systems

通过遗传密码扩增，将非天然设计氨基酸掺入活细胞和生物体中定义位点的蛋白质中，为经典方法具有挑战性或不可能解决的生物过程提供了新的见解。例如大肠杆菌基因组工程和细菌释放因子RF 1的敲除，在使琥珀抑制非常有效地用于然而，在哺乳动物细胞中将UAA有效、高蛋白产量、位点特异性地掺入蛋白质中仍然是一个突出的挑战。最近的一项研究报告了蛋白质水平的增加，表明遗传密码扩增可以针对哺乳动物细胞进行优化。3然而，到目前为止，所有将UAA定点掺入哺乳动物细胞蛋白质的系统都依赖于瞬时转染。这导致细胞之间的表达产率高度可变，限制了其用于小规模实验，不提供（或仅提供费力的）高含量（例如蛋白质组学）或筛选可能性，因此不能用于研究生物系统中的动态过程（例如分化）。为了克服这些局限性，我们建议开发一个稳定的，基因组整合系统的位点特异性引入UAA到哺乳动物细胞中的蛋白质。我们将进一步开发新的方法和生物正交化学，通过引入新的光交联部分来研究活细胞中蛋白质-蛋白质和选择性蛋白质-DNA相互作用。结合这些努力将使我们能够研究瞬时和弱蛋白质相互作用，以及在动态过程中，如细胞分化的时空定位和动力学的靶蛋白。为了实现这一点，我们的目标是（i）通过使用基于CRIPSR/Cas的基因组工程方法将吡咯赖氨酰tRNA合成酶（PylRS）/tRNA系统稳定地整合到小鼠和人类细胞的精确基因组基因座中，（ii）开发新的光诱导生物正交反应，其允许通过具有高时空分辨率的[2+2]光环加成进行特异性DNA-蛋白质交联，最后（iii）将稳定的转基因PylRS/tRNA系统与新开发的DNA特异性光交联衍生物相结合，研究DNA甲基转移酶DNMT 3B在上胚层分化过程中的时空定位、相互作用和动态。我们的研究将扩大遗传密码扩增的实用性，用于研究高度动态过程以及哺乳动物细胞中的大规模实验。我们的目标是开发新的化学将是有价值的工具，为研究的本地化和动力学的DNA相互作用的因素。

The incorporation of unnatural designer amino acids into proteins at defined sites in living cells and organisms, via genetic code expansion, has provided new insights into biological processes that are challenging or impossible to address by classical approaches.1 Recent developments, such as genome engineering in Escherichia coli and knockout of bacterial release factor RF1 have seen tremendous progress in making amber suppression very efficient for incorporation of unnatural amino acids (UAAs) in bacterial hosts.2 The efficient, high protein yielding, site-specific incorporation of UAAs into proteins in mammalian cells, represents however still an outstanding challenge. A recent study reported increased protein levels, demonstrating that genetic code expansion can be optimized for mammalian cells.3 So far, however, all systems for site-specifically incorporating UAAs into proteins in mammalian cells rely on transient transfections. This leads to highly variable expression yields between cells, limits its use to small-scale experiments, offers no (or only laborious) high content (e.g. proteomics) or screening possibilities and can thereby not be used to study dynamic processes (e.g. differentiation) in biological systems. To overcome these limitations we propose to develop a stable, genomically integrated system for the site-specific introduction of UAAs into proteins in mammalian cells. We will furthermore develop new approaches and bioorthogonal chemistries for studying protein-protein and selectively protein-DNA interactions in live cells by introducing new photo-crosslinking moieties. Combining these efforts will allow us to study transient and weak protein interactions as well as the spatio-temporal localization and kinetics of target proteins during dynamic processes such as cellular differentiation. To achieve this we aim to (i) integrate the Pyrrolysyl tRNA Synthetase (PylRS)/tRNA system stably into precise genomic loci of mouse and human cells by using a CRIPSR/Cas based genome engineering approach, (ii) develop new light induced bioorthogonal reactions that allow specific DNA-protein crosslinking via [2+2] photocycloaddition with high spatio-temporal resolution, and finally (iii) combine the stable transgenic PylRS/tRNA system with the newly developed derivatives for DNA specific photo-crosslinking to study the spatio-temporal localization, interactions and dynamics of the DNA-methyltransferase DNMT3B during epiblast differentiation. Our study will expand the utility of genetic code expansion for studies of highly dynamic processes as well as large-scale experiments in mammalian cells. The novel chemistries we aim to develop will be valuable tools for the study of localization and kinetics of DNA interacting factors.