A light-dependent aptamer-photoreceptor system enables spatiotemporal control of RNA biochemistry

Abstract

Light plays an important role in all kingdoms of life and regulates processes such as plant growth, flowering or vision in complex life forms. The growing field of optogenetics harnesses those properties by combining them into genetically encodable systems to control cellular mechanisms in a spatial and temporal manner. Several processes, such as opening of ion channels, enzymatic activity or DNA binding have been shown to be compatible with light regulation. Photoreceptor proteins, e.g., LOV (light, oxygen, voltage)-domain containing proteins are crucial in developing those processes. They are capable of reversibly changing their conformation upon blue light absorption and hence convert the incoming signal into a specific output. However, until now it was difficult to apply optogenetic control to RNA biochemistry, e.g., RNA function. <br /> Herein, a novel system was developed, combining the field of optogenetics with the aptamer technology and founding the new field of optoribogenetics. Aptamers are single stranded RNA or DNA oligonucleotides, which adapt three-dimensional structures and bind their targets with high affinity and specificity. They are chemically synthesized and generated through a process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). In this thesis, a light-dependent selection strategy termed Opto-SELEX was established, to develop aptamers specific for binding the LOV photoreceptor protein PAL, which bears a light-activatable RNA binding domain. This strategy yielded two RNA aptamer motifs termed motif 1 and motif 2, which selectively interact with PAL in its light conformation. Both aptamers were truncated to 17 and 19 nucleotide long RNA hairpins, respectively, and exhibit affinities of approximately 100 nM towards the light conformation of PAL. <br /> In-deep analysis of the aptamers revealed that motif 1 displayed reduced binding at increased temperatures and mutations in the loop regions led to abolished binding. In contrast, motif 2 lost binding affinity in absence of Mg²⁺ ions and was more prone to mutations in the stem regions. <br /> This knowledge was applied in the design of conjoint aptamer-mRNA molecules that were capable of regulating gene expression on RNA level in a light-dependent manner. Whereas translation of a luciferase reporter was unaffected by PAL in darkness, translation was decreased to 15% for motif 2 and 55% for motif 1 after irradiation with blue light. <br /> Finally, the obtained aptamers were utilized to identify putative native targets in the bacterial Nakamurella multipartita genome, the organism PAL originates from. <br /> In summary, this thesis demonstrates the successful development of a light-dependent RNA-aptamer-photoreceptor system, which is capable of controlling RNA function in vitro and in mammalian cells. This optoribogenetic system is small and modular and offers various possibilities for future applications, e.g., to control RNA function in vivo or incorporation of the aptamer hairpins into new systems to regulate RNA.