Characterizing the Use of the RNA Mango Aptamer for RNA Pull‐Downs and Single Molecule Fluorescence

Abstract

Methods for investigating the conformation and interactions of RNA and RNA/protein complexes are currently limited. This in turn has restricted our ability to understand the dynamics and interactions of small nuclear RNAs of the spliceosome. One way to address this challenge is by “tagging” the RNAs with an aptamer so that they can be readily purified and visualized. RNA Mango is an aptamer that has a high affinity (KD<10 nM) and specificity for derivatives of its fluorescent ligand, thiazole orange (TO1). Fluorescence of TO1 is enhanced 103‐fold when it is bound to the parallel‐stranded G‐quandruplex of RNA Mango, making the use of this ligand‐aptamer pair a promising strategy in RNA localization and purification experiments1. What makes this aptamer truly exciting is its ability to enable studies by a variety of biochemical techniques, including single molecule Förster Resonance Energy Transfer (smFRET) experiments and pull‐down assays. The goal of this work was to explore the versatility of RNA Mango incorporation into snRNAs through such experiments.For smFRET experiments, we labeled distinct positions of RNA Mango with Cy5 and Cy3 fluorophores, and determined that the aptamer has a folded FRET state of ~0.7 in monovalent ionic buffer solutions. Additional smFRET experiments suggest that the presence of TO1 derivatives in solution do not affect the folding of RNA Mango, whose properly folded G‐quadruplex is critical for the fluorescence of TO1. Thus, the RNA Mango aptamer itself is stable under a variety of conditions and TO1 binding may only introduce small conformational changes.We created a Saccharomyces cerevisiae strain with RNA Mango incorporated into the U4 snRNA, a component of the spliceosome, such that RNA Mango and TO1 derivatives can be used to isolate U4 and U4 containing splicing complexes. Primer extension assays comparing S. cerevisiae with wildtype and U4‐Mango showed that RNA Mango was successfully incorporated into the genome of our modified strain. We were able to purify snRNP complexes using U4 snRNA:RNA Mango and biotinylated TO1. This work demonstrates that RNA Mango can be integrated into the U4 snRNP to give further insights into the complexities of interactions in the spliceosome.Support or Funding InformationAaron Hoskins is a Beckman Young Investigator of the Arnold and Mable Beckman Foundation