UC San Diego

RNA is one of the most important biomacromolecules in the living systems, manipulating a highly complexed collection of functions which are critical to the regulation of numerous cellular processes. In the past few years, our lab has developed a versatile and powerful RNA modifying technique, named RNA-TAG. The RNA-TAG technique utilizes a bacterial tRNA guanine transglycosylase (TGT) to exchange a guanine nucleobase within a specific 17-nucleotide motif (Tag) for synthetic pre-queuosine1 (preQ1) derivatives. By inserting this Tag sequence into an RNA of interest, we can covalently and site-specifically conjugate functional small-molecules onto any RNA of interest. I applied our RNA-TAG technique to manipulate the functions of different types of cellular RNA, for example, mRNA for translation regulation and the singe guide RNA (sgRNA) for CRISPR/Cas9 gene editing.To interfere with the translation process of mature mRNA, I systematically conjugated three bulky visible-light photocleavable caging groups along the 5’ untranslated region (5’-UTR) of an mRNA, severely reducing its translation activity. Upon visible-light photocleavage of these caging groups, mRNA translation was resorted. To expand our technology, we further designed two photo-sensitive caging groups which can be sequentially cleaved by two wavelength of lights (405 nm and 488 nm). In this manner, a sequential/multiplexed photo-activation of two mRNAs within the same cells was achieved. Based on our RNA-TAG technique, I developed a novel technique named RNA-CLAMP, which can covalently and site-specifically crosslink (‘clamp’) two internal stem-loops within a RNA of interest. By designing small-molecule substrates which contain a cleavable linker and two preQ1 moieties, I achieved accurate ‘clamping’ and rapid release of the target RNA, which significantly alter its secondary/ternary structure, resulting in gain/loss of functions. I applied the RNA-CLAMP technique to the sgRNA of the CRISPR/Cas9 gene editing system. Upon ‘clamping’ of two internal loops (the tetra-loop and stem-loop2) of the sgRNA, the Cas9 gene editing system was completely deactivated. However, the cleavage of the crosslinker with an external stimulus, for example, light irradiation, released the sgRNA and fully activated gene editing in live mammalian cells. To the best of knowledge, this CRISPR/Cas9 gene editing photo-regulation technology offers the best dynamic range, flexibility, multiplexing capability and accessibility to date. We believe with further development, the RNA-CLAMP technique will have tremendous applications in manipulating other types of RNAs, such as ribozymes, microRNAs, and long non-coding RNAs. Such techniques will serve as powerful tools in studying complex cellular networks as well as developing novel therapeutics applications.