Development of a novel method for tracking of circular RNAs in living cellsMlakar, Tjaša (Avtor) Murn, Jernej Murn (Univ. California, Riverside) (Mentor) Režen, Tadeja (Komentor) circRNARNA aptamerPepperfluorescent taggingmethod developmentCircular RNAs (circRNA) are generated during a non-canonical splicing event called backsplicing. It was long thought that these transcripts are only noise produced by aberrant splicing events and were therefore quickly discarded. In recent years however, with the advancement of technology, several circRNAs have been identified in humans and other organisms. These results have led researchers to question the actual role of circRNAs in cell biology. So far, several circular transcripts have been implied to play a part in different human diseases, including neurological disorders, cancer, and cardiovascular diseases. Their potential roles have also led circRNAs to be researched in terms of clinical applications, including their use as biomarkers and therapeutics. Thus far, only a minority of circRNAs identified with RNA sequencing have been sufficiently investigated. There are several methods currently in use to investigate circRNAs, including fluorescence in situ hybridization (FISH). With the numerous drawbacks of RNA-FISH, the need has arisen for a novel method for circRNA investigation in living cells. We developed a novel method that allows fluorescent labeling of circRNAs, but not linear RNAs, with known fluorogenic RNA aptamers (Pepper). These aptamers were halved and added to each side of the linear transcripts and only formed a functional RNA aptamer when the backsplice junction (BSJ) was formed. With the addition of the HBC fluorescence dye, which is able to incorporate into the RNA aptamer structure, the circRNA transcripts were visualized under fluorescence microscope. The novel method overcomes the drawbacks of FISH and allows the investigation of circRNAs in living cells. We demonstrated that the split RNA aptamer does not interfere with circRNA circularization and the formation of a BSJ region. We also demonstrated that a split RNA aptamer is able to form a whole and is also functional when adding HBC dye. In addition, the method has the potential to be used to track circRNA in living cells with sufficient optimization. All in all, the development of a novel method potentially enables a more efficient investigation of circRNAs in living cells.20222022-09-13 08:15:00Magistrsko delo/naloga140184sl