3′-end fluorescence labelling of long RNAs using terminal deoxynucleotidyl transferase

Abstract: Through recent years, RNA has rapidly gone from mainly being known as a passive information-carrying molecule within the central dogma of molecular biology to becoming the active ingredient in multiple therapeutics, such as in vaccines and potential drugs against cancer. However, one of the main challenges with RNA as a therapeutic is efficient delivery to cells. Understanding the mechanism through which RNA enters cells is therefore crucial to develop more effective RNA therapeutics. New methods to accurately study RNA in cells are becoming increasingly important to fully understand these processes. Fluorescence is one such method commonly used to analyse RNA in cellulo. Since natural nucleotides are virtually non-fluorescent, fluorescent labels have to be introduced to enable analysis. tCO is a fluorescent cytosine analogue causing little perturbance to the RNA’s structure and function. This thesis explores a novel way of enzymatically labelling RNA with tCO using terminal deoxynucleotidyl transferase (TdT), a DNA polymerase capable of template-independent 3′-extension of nucleic acids. RNA coding for enhanced green fluorescent protein (eGFP) was produced through in vitro transcription. These strands were then extended using the TdT enzyme to add a mixture of natural nucleotide triphosphates (NTPs) and tCO-triphosphate (tCOTP) to their 3′-ends. Reactions using varying tCOTP /canonical NTPs-ratios, different cofactors, as well as different cofactor concentrations were performed to study their effect on the labelling performance. The RNA extension was evaluated using gel electrophoresis, as well as UV-vis absorption, excitation, and emission spectroscopy. tCO was successfully added to the 3′-ends of the RNA. The efficiency of the labelling seemed to be dependent on the available tCOTP amount, the used cofactor, the cofactor concentration, and the RNA sequence. Altogether, TdT labelling of RNA was demonstrated as a feasible way of labelling long, functional RNAs with tCO.