信使核糖核酸
荧光素酶
非翻译区
转染
翻译(生物学)
分子生物学
三素数非翻译区
五素未翻译区
核苷酸
细胞生物学
化学
平动调节
核糖核酸
生物
蛋白质生物合成
生物化学
基因
作者
Željka Trepotec,Manish Kumar Aneja,Johannes Geiger,Günther Hasenpusch,Christian Paar,Carsten Rudolph
出处
期刊:Tissue Engineering Part A
[Mary Ann Liebert]
日期:2019-01-01
卷期号:25 (1-2): 69-79
被引量:41
标识
DOI:10.1089/ten.tea.2017.0485
摘要
The 5'-untranslated region (5'-UTR) of mRNA contains structural elements, which are recognized by cell-specific RNA-binding proteins, thereby affecting the translation of the molecule. The activation of an innate immune response upon transfection of mRNA into cells is reduced when the mRNA comprises chemically modified nucleotides, putatively by altering the secondary structure of the molecule. Such alteration in the 5'-UTR in turn may affect the functionality of mRNA. In this study, we report on the impact of seven synthetic minimalistic 5'-UTR sequences on the translation of luciferase-encoding unmodified and different chemically modified mRNAs upon transfection in cell culture and in vivo. One minimalistic 5'-UTR, consisting of 14 nucleotides combining the T7 promoter with a Kozak consensus sequence, yielded similar or even higher expression than a 37 nucleotides human alpha-globin 5'-UTR containing mRNA in HepG2 and A549 cells. Furthermore, also the kind of modified nucleotides used in in vitro transcription, affected mRNA translation when using different translation regulators (Kozak vs. translation initiator of short UTRs). The in vitro data were confirmed by bioluminescence imaging of expression in mouse livers, 6 h postintravenous injection of a lipidoid nanoparticle-formulated RNA in female Balb/c mice. Luciferase measurements from liver and spleen showed that minimal 5'-UTRs (3 and 7) were either equally effective or better than human alpha-globin 5'-UTR. These findings were confirmed with a human erythropoietin (hEPO)-encoding mRNA. Significantly, higher levels of hEPO could be quantified in supernatants from A549 cells transfected with minimal 5'-UTR7 containing RNA when compared to commonly used benchmarks 5'-UTRs. Our results demonstrate the superior potential of synthetic minimalistic 5'-UTRs for use in transcript therapies.
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