Development of fluorogenic RNA aptamers for cellular imaging of RNA and genomic loci

Abstract

In recent years, there has been an explosion of SELEX-evolved fluorescent RNA aptamers, such as Spinach, Broccoli, Corn and Mango. Fluorogenic RNA aptamers have sparked a lot of interest and hold great potential to enable background-free visualisation of RNA molecules in cellular environments. However, their application has been limited by relatively inefficient folding in vivo and fluorescent stability. Therefore, evolving new RNA aptamers with improved brightness and stability should better their use in cellular imaging. Three new Mango-based aptamers have recently been selected from the original Mango RNA SELEX pool using microfluidic- assisted in vitro compartmentalization and fluorescence-activated sorting. This thesis demonstrates the use of these new aptamer variants to image small non-coding RNAs (such as 5S rRNA, U6 snRNA and mgU2-47 scaRNA) in both fixed and live human cells with improved sensitivity and resolution. Upon expression the modified RNAs subcellular localisation pattern is conserved, as validated using immunofluoresence. Recent work with tandem Mango arrays shows increased sensitivity, which enables the visualization of single mRNA molecules in live and fixed cells. Furthermore, it is shown that the tandem Mango arrays don’t affect the expected localization of a cytoplasmic mRNA (β-actin) and the nuclear long non- coding RNA (NEAT-1). Furthermore, these RNA aptamers can also be used to label genomic loci via CRISPR/Cas9 mediated genome targeting with improved contrast. This allows for the targeting of short genomic repeats in a less invasive manner with regards to current methodologies. Taken together this data shows that new Mango aptamers are vastly improved for cellular imaging over previous RNA aptamers, and can in principle be incorporated into a wide range of coding and non-coding RNAs.