Stability analysis of chemically modified mRNA using micropattern-based single-cell arrays

Ferizi, Mehrije; Leonhardt, Carolin; Meggle, Christian; Aneja, Manish K.; Rudolph, Carsten; Plank, Christian und Rädler, Joachim O. (2015): Stability analysis of chemically modified mRNA using micropattern-based single-cell arrays. In: Lab on a Chip, Bd. 15, Nr. 17: S. 3561-3571

Abstract

The measurement of mRNA turnover in living cells plays an important role in the search for stable mRNA constructs for RNA-based therapies. Here we show that automated time-lapse microscopy combined with micropatterned arrays allows for efficient high-throughput monitoring of fluorescent reporter protein expression at the single-cell level. The fluorescence time courses after mRNA transfection yield the distribution of individual mRNA expression and degradation rates within a population. We compare mRNA constructs with combinations of 5′ and 3′ UTR sequences and find a systematic broadening and shift towards longer functional half-lives for UTR stabilized mRNA. At the same time the life time distribution of the destabilized EGFP reporter protein was found to be constant and narrowly distributed. Using mathematical modeling, we show that mRNA functional life-time predicts the time-integrated protein level, i.e. the area under the curve (AUC) of mRNA translation. Our approach paves the way for quantitative assessment of hitherto unexplored mRNA functional life time heterogeneity, possibly predicated on multiple mRNA secondary structures and its dependence on UTR sequences.

• Dokumententyp: Zeitschriftenartikel
• Publikationsform: Publisher's Version
• Fakultät: Physik
• Themengebiete: 500 Naturwissenschaften und Mathematik > 530 Physik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• ISSN: 1473-0197
• Allianz-/Nationallizenz: Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
• Sprache: Englisch