Development of chemical tools for imaging RNA and studying RNA and protein interactions

Tools for study of RNA are divided into two groups: RNA imaging and RNA crosslinking (Chapter 1). RNA imaging denotes visualization of target RNA by labelling it with fluorophores. RNA crosslinking refers to investigating the function of target RNA by capturing bio-macromolecules the RNA interacts with.

In consideration of low abundance of some RNAs in living cells, in chapter 2, we have synthesized a new RNA probe based on previously reported aptamers. which reduces background signal to a large extent. We realized imaging of the RNA aptamer in mammalian cells, and the fluorescence obtained from newly constructed probe was apparently brighter.

In chapter 3, we developed two psoralen based length tunable crosslinkers (AMT-NHS and AMT dimer) for studying RNA-RNA interactions or RNA-protein interactions. We have synthesized the crosslinkers and verified in vitro experiment that RNA-RNA interactions could be effectively captured by the AMT dimer, and RNA-protein interactions could be effectively tracked by AMT-NHS. In chapter 4, we proved that AMT-NHS could be applied to study RNA-protein interactions in living cells. We also proved that AMT-NHS CLIP is a stable and efficient method for studying RNA-protein interactions, it captured a large portion of interactions that traditional CLIP method may miss.

In chapter 5, we have synthesized and incorporated an unnatural photoactivatable amino acid into a protein on a specific site by expanding genetic code, which paves the way to develop a novel and powerful CLIP method to capture more comprehensively RNA-protein interactions.