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.

Popular Abstract

Every living cell making up the human body carries a copy of the genome. This is essentially the recipe for how to make a human. The recipe is written as long strands of the four letters A, C, G, and T, collectively known as nucleotides, spelling out genes as long strands of DNA. Genes get read by the cell and copies are made in the form of ribonucleic acids (RNAs) with the letter T replaced with U. The RNA can then be translated by the protein factory of the cell, the ribosome, into a functioning protein, able to perform the actions needed by the cell. This is the central dogma of molecular biology. A big field of research today is how to use RNA in new medical therapies, as was done for the vaccines used against the Covid-19 virus. However, delivering therapeutic RNA to cells remains a challenge. New techniques to study RNA in cells are therefore becoming increasingly important. One such technique is the use of fluorescence. Fluorophores are molecules that, when exposed to photons of the right energy level, emit new photons with lower energy which can be detected. The process where a fluorophore emits a photon is called fluorescence and allows for highly sensitive analysis. As the RNA building blocks are virtually non-fluorescent themselves, fluorescent labels have to be attached to the RNA to enable analysis.

This thesis explores the use of the enzyme terminal nucleotide transferase (TdT) to label the ends of long RNA strands using the fluorescent label tCO. Conventional fluorescent labels are generally large and bulky, leading to possible adverse effects on the structure and function of the labelled RNA. tCO is a fluorescent label that has been designed to replace and act as the natural C nucleotide within the RNA sequence, making it a less perturbing label for studying the native behaviour of RNA with fluorescence. To attach tCO to RNA, the enzyme TdT is used. TdT is an enzyme that has the unique ability to extend nucleic acids, such as RNA, by taking the precursors to nucleotides, nucleoside triphosphates (tCO-triphosphate in the case of tCO), and adding them to one of the RNA ends. To work, TdT requires a metal ion present in the reaction called the cofactor. Reactions using different reaction conditions were performed to study their effect on the incorporation of tCO.

TdT was successfully used in this thesis to label RNA using tCO. The amount of incorporated tCO was shown to be dependent on the amount of available tCO triphosphate, the cofactor used, and the concentration of the cofactor. RNA sequence also seemed to affect the incorporation of tCO. The number of tCOs attached to the RNA strands was considerably less than what has previously been done with shorter RNA.

• author: Lidberg, Johannes
• supervisor: Marcus Wilhelmsson
• organization: Department of Chemistry
• course: KEMK10 20222
• year: 2022
• type: M2 - Bachelor Degree
• subject: Chemistry
• keywords: Physical biochemistry, Fluorescence, In vitro transcription, RNA, Terminal deoxynucleotidyl transferase, Nucleobase analogue
• language: English
• id: 9103613
• date added to LUP: 2022-12-06 14:11:00
• date last changed: 2022-12-06 14:11:00