anti-reverse cap analogue  (TriLink)

Journal: Frontiers in Pharmacology

Article Title: PepFect14 mediates the delivery of mRNA into human primary keratinocytes and in vivo

doi: 10.3389/fphar.2023.1219761

Figure Lengend Snippet: PF14-mRNA nanoparticles are with suitable characteristics for cellular delivery. ( A,B ) For DLS, nanoparticles were prepared using PF14 and four different mRNAs: mCherry, luciferase, EGFP and Cy5-mRNA at charge ratio (CR) 2:1. If indicated, nanoparticles were supplemented with PS80, chloroquine, MgCl 2 and/or CaCl 2 . The white bars represent the treatments, where only PF14 or indicated mRNAs were added to the cells. Data are representative of three technical replicates and expressed as ± SEM, n = 3, one-way ANOVA with post-hoc Šidák test was used, ns – not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: mRNAs: Anti-reverse cap analogue (ARCA) mRNA encoding enhanced green fluorescent protein (EGFP), i.e., EGFP-mRNA (5-moUTP), ARCA Cy5-labeled EGFP-mRNA (5mo-UTP), and CleanCap mCherry-mRNA (5mo-UTP) were purchased from APExBIO Technology (Houston, TX, USA), and CleanCap Fluc-mRNA, i.e., luciferase-mRNA (5moU) from TriLink Biotechnologies (San Diego, CA, USA).

Techniques: Luciferase

Journal: Nucleic Acids Research

Article Title: Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons

doi: 10.1093/nar/gkl442

Figure Lengend Snippet: Characterization of the T7 RNAP mRNA/pT7Luc expression system. ( A ) A549 (white bars) and PC-3 (grey bars) cells were transfected with 400 ng pT7Luc with 100 ng T7 RNAP mRNA modified with either m7G(5′)ppp(5′)G (Cap), an anti-reverse cap analogue (ARCA), a poly(A64) tail or a poly(A) tail of ∼200 residues (A200) as indicated. ( B ) A549 cells were transfected with 500 ng pT7Luc (time 0 h) prior to transfection 12 h later with 125 ng T7 RNAP mRNA (time 12 h), or vice versa as indicated. Cells were lysed after a further 12 h and luciferase activity measured. In control experiments, A549 cells were transfected with both pT7Luc and T7 RNAP mRNA at time 0 h, and lysed 12 h later. ( C ) A549 cells were transfected with 400 ng pT7Luc and 100 ng T7 RNAP mRNA, and at the indicated time point lysed and total RNA extracted. A qRT–PCR assay was used to quantify the amount of T7 RNAP mRNA present (pg) as described in Materials and Methods. ( D ) A549 cells were incubated in the presence of DOTAP alone, or the indicated nucleic acid complex for 4 h in serum-free media. The media was discarded and replaced with fresh media containing 10% FCS and the MTS assay used to assess cellular viability after 24 h. For all transfections 3.5 × 10 4 cells were plated per well 24 h prior to transfection and DOTAP used at a (w/w) ratio of five relative to the nucleic acid component. At 24 h post-transfection, unless otherwise indicated, cells were lysed and luciferase activity measured using the Victor 2 multilabel counter. Results are shown as a mean and SD from at least three samples. ( *** P < 0.0001).

Article Snippet: In vitro transcription to produce capped mRNA encoding T7 RNAP protein was performed using the Ribomax™ large scale mRNA production system (Promega, Southampton, UK), with the addition of either a standard m7G(5′)ppp(5′)G cap, or anti-reverse cap analogue (Ambion) substituted for a portion of the rGTP at a 4:1 ratio, as recommended by the manufacturer.

Techniques: Expressing, Transfection, Modification, Luciferase, Activity Assay, Quantitative RT-PCR, Incubation, MTS Assay