Enlightening RNA biology – Insights from Fluorescent Nucleobase Analogues
Doctoral thesis, 2025
Well-characterized, native-like probes to study RNA and its nucleotides are essential for advancing both fundamental research and the development of RNA-based therapeutics. Fluorescent nucleobase analogues (FBAs) offer unique opportunities to probe nucleic acids under physiological conditions while preserving native interaction patterns. This thesis explores the use of fluorescent nucleosides to investigate RNA and nucleotide metabolism, with a particular focus on live-cell imaging applications. It includes a comprehensive photophysical characterization of FBAs, emphasizing their suitability for various microscopy techniques. These insights are applied to monitor the spontaneous cellular uptake of FBAs using fluorescence microscopy, including confocal imaging, two-photon excitation microscopy, and fluorescence lifetime imaging. One adenine analogue, 2CNqA, emerged as a key compound due to its ability to enter cells and localize to the nucleus, i.e. the site of RNA synthesis. In addition to imaging, techniques such as flow cytometry and spectroscopy were employed to elucidate the uptake pathways and the metabolic processing of 2CNqA, including its incorporation into RNA. This work also demonstrates the incorporation of 2CNqA into long RNA via in vitro transcription, including a thorough characterization of 2CNqA incorporation degree and its photophysical properties in an RNA environment. The subsequent cellular delivery of this FBA-labelled mRNA and the expression of the encoded fluorescent protein are investigated for applicability for RNA studies in living cells.
Together, the thorough characterization of FBAs, the observed spontaneous uptake and subsequent RNA labelling, and the in vitro transcription-based labelling strategy constitute a versatile toolbox for studying dynamic aspects of RNA biology and RNA-based drug delivery in living cells. This thesis also highlights the potential of diverse readout methods for advancing RNA imaging and analysis.
Together, the thorough characterization of FBAs, the observed spontaneous uptake and subsequent RNA labelling, and the in vitro transcription-based labelling strategy constitute a versatile toolbox for studying dynamic aspects of RNA biology and RNA-based drug delivery in living cells. This thesis also highlights the potential of diverse readout methods for advancing RNA imaging and analysis.
Fluorescent base analogues
RNA
nucleotides
fluorescence microscopy
live cell imaging
fluorescence
spectroscopy
in vitro transcription
RNA building blocks
nucleosides
HA4, Hörsalsvägen 4
Opponent: Prof. Dr. Christian Eggeling Friedrich Schiller University, Jena, Germany.
Author
Pauline Pfeiffer
Chemistry and Biochemistry Phd Students and Postdocs
Multiphoton characterization and live cell imaging using fluorescent adenine analogue 2CNqA
Physical Chemistry Chemical Physics,;Vol. 25(2023)p. 20218-20224
Journal article
Metabolic RNA labeling in non-engineered cells following spontaneous uptake of fluorescent nucleoside phosphate analogues
Nucleic Acids Research,;Vol. 52(2024)p. 10102-10118
Journal article
Expanding fluorescent base analogue labelling of long RNA by in vitro transcription; Pauline PfeifferŦ, Alma F. E. KarlssonŦ, Jesper R. Nilsson, L. Marcus Wilhelmsson
Monitoring Nucleoside Metabolism in Living Cells with a Nucleobase Analogue via Fluorescence Lifetime Imaging; Pauline Pfeiffer, Niusha Bagheri, Chen Qian, Jerker Widengren, L. Marcus Wilhelmsson
Increased Brightness of Fluorescent Uridine qU inside Single- and Double-Stranded RNA; Alma F. E. KarlssonŦ, Pauline PfeifferŦ, Hoang-Ngoan Le, Tom Baladi, Anders Dahlén, L. Marcus Wilhelmsson
RNA är en central molekyl i celler som bland annat styr hur proteiner bildas och den är därför en nyckelspelare i cellens maskineri och ligger till grund för flera nya läkemedel, såsom mRNA-vacciner. För att förstå RNA:s funktioner bättre – både inom grundforskning och vid utveckling av nya läkemedel – behövs verktyg som gör det möjligt att följa RNA i levande celler. I detta arbete har fluorescerande byggstenar, så kallade fluorescerande basanaloger (FBAer), studerats. Dessa molekyler liknar RNA:s naturliga byggstenar men har den unika egenskapen att de kan lysa, vilket gör dem synliga med hjälp av avancerad mikroskopi. Avhandlingen visar att FBAer kan fungera som verktyg för att studera RNA-omsättning i celler i realtid på ett nytt sätt.
En av de molekyler som vi har utvecklat, 2CNqA, hamnade i fokus eftersom den visade särskilt lovande egenskaper genom att kunna ta sig in i celler och nå cellkärnan, där RNA bildas. Med hjälp av tekniker som mikroskopi, flödescytometri och spektroskopi har upptag, transport och insättning av 2CNqA i RNA undersökts. Dessutom har 2CNqA använts för att märka långa RNA-molekyler, som framställts utanför celler m.h.a. så kallad in vitro transkription och sedan levererats till celler för att följa RNA:t, och se bildning av fluorescerande proteiner som RNA:t kodar för.
Resultaten bidrar till forskningen kring RNA och utvecklingen av framtidens RNA-baserade läkemedel genom att studera hur dessa transporteras och verkar i levande celler. Arbetet lyfter fram olika metoder för att läsa av och analysera dessa processer.
En av de molekyler som vi har utvecklat, 2CNqA, hamnade i fokus eftersom den visade särskilt lovande egenskaper genom att kunna ta sig in i celler och nå cellkärnan, där RNA bildas. Med hjälp av tekniker som mikroskopi, flödescytometri och spektroskopi har upptag, transport och insättning av 2CNqA i RNA undersökts. Dessutom har 2CNqA använts för att märka långa RNA-molekyler, som framställts utanför celler m.h.a. så kallad in vitro transkription och sedan levererats till celler för att följa RNA:t, och se bildning av fluorescerande proteiner som RNA:t kodar för.
Resultaten bidrar till forskningen kring RNA och utvecklingen av framtidens RNA-baserade läkemedel genom att studera hur dessa transporteras och verkar i levande celler. Arbetet lyfter fram olika metoder för att läsa av och analysera dessa processer.
RNA is a key molecule in cells that controls, for example, how proteins are made. Because of this central role, RNA is not only a key player in the cell's machinery but also forms the basis of several modern medicines, such as mRNA vaccines. To better understand the functions of RNA – both in basic research and in the development of new drugs – tools are needed to track and study RNA in living cells. In this work, fluorescent building blocks, known as fluorescent base analogues (FBAs), have been studied. These molecules are like natural building blocks of RNA but have the unique property of being able to glow (fluoresce), making them visible using advanced microscopy. The thesis shows that FBAs can serve as tools to study RNA turnover in cells in real time.
One of the molecules that we have developed, 2CNqA, was particularly interesting because it is able to enter cells and reach the nucleus, where RNA is formed. Using techniques such as microscopy, flow cytometry and spectroscopy, the uptake, transport and incorporation of 2CNqA into RNA have been investigated. In addition, 2CNqA has been used to label long RNA molecules produced outside cells by in vitro transcription. This RNA is then delivered into cells, where it can be tracked due to its fluorescent properties and where the formation of fluorescent proteins encoded by the RNA can be observed.
The results of this thesis contribute to RNA research and the development of future RNA-based therapeutics by studying how they are transported and act in living cells. The work highlights different methods for studying and analysing these processes.
One of the molecules that we have developed, 2CNqA, was particularly interesting because it is able to enter cells and reach the nucleus, where RNA is formed. Using techniques such as microscopy, flow cytometry and spectroscopy, the uptake, transport and incorporation of 2CNqA into RNA have been investigated. In addition, 2CNqA has been used to label long RNA molecules produced outside cells by in vitro transcription. This RNA is then delivered into cells, where it can be tracked due to its fluorescent properties and where the formation of fluorescent proteins encoded by the RNA can be observed.
The results of this thesis contribute to RNA research and the development of future RNA-based therapeutics by studying how they are transported and act in living cells. The work highlights different methods for studying and analysing these processes.
Subject Categories (SSIF 2025)
Molecular Biology
Organic Chemistry
Physical Chemistry
ISBN
978-91-8103-269-7
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5727
Publisher
Chalmers
HA4, Hörsalsvägen 4
Opponent: Prof. Dr. Christian Eggeling Friedrich Schiller University, Jena, Germany.