Metabolic RNA labeling in non-engineered cells following spontaneous uptake of fluorescent nucleoside phosphate analogues
Journal article, 2024
RNA and its building blocks play central roles in biology and have become increasingly important as therapeutic agents and targets. Hence, probing and understanding their dynamics in cells is important. Fluorescence microscopy offers live-cell spatiotemporal monitoring but requires labels. We present two fluorescent adenine analogue nucleoside phosphates which show spontaneous uptake and accumulation in cultured human cells, likely via nucleoside transporters, and show their potential utilization as cellular RNA labels. Upon uptake, one nucleotide analogue, 2CNqAXP, localizes to the cytosol and the nucleus. We show that it could then be incorporated into de novo synthesized cellular RNA, i.e. it was possible to achieve metabolic fluorescence RNA labeling without using genetic engineering to enhance incorporation, uptake-promoting strategies, or post-labeling through bio-orthogonal chemistries. By contrast, another nucleotide analogue, pAXP, only accumulated outside of the nucleus and was rapidly excreted. Consequently, this analogue did not incorporate into RNA. This difference in subcellular accumulation and retention results from a minor change in nucleobase chemical structure. This demonstrates the importance of careful design of nucleoside-based drugs, e.g. antivirals to direct their subcellular localization, and shows the potential of fine-tuning fluorescent base analogue structures to enhance the understanding of the function of such drugs.
Author
Pauline Pfeiffer
Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry
Jesper Nilsson
Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry
Audrey Gallud
AstraZeneca AB
Chalmers, Life Sciences, Chemical Biology
Tom Baladi
Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry
AstraZeneca AB
Hoang Ngoan Le
AstraZeneca AB
Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry
Mattias Bood
University of Gothenburg
AstraZeneca AB
Malin Lemurell
AstraZeneca AB
Anders Dahlén
AstraZeneca AB
Morten Grötli
University of Gothenburg
Elin Esbjörner Winters
Chalmers, Life Sciences, Chemical Biology
Marcus Wilhelmsson
Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry
Nucleic Acids Research
0305-1048 (ISSN) 1362-4962 (eISSN)
Vol. 52 17 10102-10118Funktionell leverans av nukleotid-baserade läkemedel
Swedish Foundation for Strategic Research (SSF) (IRC15-0065), 2017-03-01 -- 2024-12-31.
Endogenous non-invasive fluorescence labeling of RNA and its use in cell imaging
Swedish Research Council (VR) (2021-04409), 2021-12-01 -- 2025-11-30.
Subject Categories
Biochemistry and Molecular Biology
DOI
10.1093/nar/gkae722
PubMed
39162218