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

通过基因密码扩展，在活细胞和生物体的特定位置将非天然设计氨基酸掺入蛋白质中，为用经典方法解决具有挑战性或不可能解决的生物过程提供了新的见解。1最近的发展，如大肠杆菌基因组工程和敲除细菌释放因子RF1，在使琥珀抑制非常有效地在细菌宿主中掺入非天然氨基酸(UaAs)方面取得了巨大进展。2然而，将UaA高效、高蛋白产量、特定部位地掺入哺乳动物细胞的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，表明遗传密码的扩展可以对哺乳动物细胞进行优化。3然而，到目前为止，所有在哺乳动物细胞中将尿苷酸定点整合到蛋白质中的系统都依赖于瞬时转基因。这导致细胞之间的表达产量高度不同，限制了其在小规模实验中的使用，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)。为了克服这些限制，我们建议开发一种稳定的、基因组集成的系统，用于将尿氨酸引入哺乳动物细胞中的蛋白质中。我们将进一步开发新的方法和生物正交化学来研究活细胞中蛋白质-蛋白质和蛋白质-DNA的选择性相互作用，通过引入新的光交联体。结合这些努力将使我们能够研究瞬时和微弱的蛋白质相互作用，以及在细胞分化等动态过程中目标蛋白质的时空定位和动力学。为了实现这一目标，我们的目标是(I)通过基于CRIPSR/Cas的基因组工程方法将吡咯酰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位置上；(Ii)开发新的光诱导生物正交反应，使其能够通过[2+2]光环加成进行高时空分辨率的DNA-蛋白质交联；(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Dnmt3b在上皮细胞分化过程中的时空定位、相互作用和动力学。我们的研究将扩大遗传密码扩展的用途，用于研究高度动态的过程以及在哺乳动物细胞中进行大规模实验。我们致力于开发的新型化学手段将成为研究DNA相互作用因子的定位和动力学的有价值的工具。

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.