Authors
Rik Oude Egberink1, Roland Brock1, Marco G. Drexelius2, Félix Gayraud2, Ines
Neundorf2
Department
- Dept. of Medical BioSciences, Radboud university medical center, Nijmegen, The Netherlands,
- Marco G. Drexelius, Félix Gayraud, Ines Neundorf, Institute of Biochemistry, Dept. of Chemistry, University of Cologne, Cologne, Germany.
Introduction
The clinical application of mRNA critically depends on delivery vehicles for
efficient packaging and intracellular delivery. In the development of novel
delivery vehicles both the capacity to induce cellular uptake and to enable mRNA
expression are crucial. Important, in many cases, uptake does not correlate with
activity as delivery vehicles may get captured inside the endolysosomal
compartment due to poor endosomal release. Ideally, a reporter mRNA should
enable detection of trafficking and delivery simultaneously. However, the
attachment of fluorophores to nucleobases negatively impacts translation
efficiency. As a consequence, for previously available fluorescently labelled
mRNAs activity could only be seen when using highly active delivery vehicles,
such as lipofectamine. Here, we show that a novel, highly active fluorescently
labelled mRNA encoding for secreted nanoluciferase (SecNLuc) enables
detection of cellular uptake and subcellular distribution by confocal microscopy
in one experiment together with determination of expression by luciferase
activity also for delivery systems with intermediate activity. Use of this mRNA
greatly enhances the information content of experiments aiming at
understanding of delivery processes and the development of new delivery
vectors.
