title: Stability analysis of chemically modified mRNA using micropattern-based single-cell arrays.
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.
authors.affiliation: Institute of Molecular Immunology - Experimental Oncology, Technische Universität München, Munich, 81675 Germany.
authors.forename: Mehrije
authors.initials: M
authors.lastname: Ferizi
authors.forename: Carolin
authors.initials: C
authors.lastname: Leonhardt
authors.forename: Christian
authors.initials: C
authors.lastname: Meggle
authors.forename: Manish K
authors.initials: MK
authors.lastname: Aneja
authors.forename: Carsten
authors.initials: C
authors.lastname: Rudolph
authors.forename: Christian
authors.initials: C
authors.lastname: Plank
authors.forename: Joachim O
authors.initials: JO
authors.lastname: Rädler
mesh_terms: D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry*; D059010:Single-Cell Analysis / Q000379:methods*; D046888:Tissue Array Analysis / Q000379:methods*; D014162:Transfection
chemical_list: D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins