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. 2003 Sep;9(9):1108-22.
doi: 10.1261/rna.5430403.

Novel "anti-reverse" cap analogs with superior translational properties

Jacek Jemielity  1 , Tolvert Fowler, Joanna Zuberek, Janusz Stepinski, Magdalena Lewdorowicz, Anna Niedzwiecka, Ryszard Stolarski, Edward Darzynkiewicz, Robert E Rhoads
Affiliations

Novel "anti-reverse" cap analogs with superior translational properties

Jacek Jemielity et al. RNA. 2003 Sep.

Abstract

Synthetic analogs of the 5'-terminal caps of eukaryotic mRNAs and snRNAs are used in elucidating such physiological processes as mRNA translation, pre-mRNA splicing, intracellular transport of mRNA and snRNAs, and mRNA turnover. Particularly useful are RNAs capped with synthetic analogs, which are produced by in vitro transcription of a DNA template using a bacteriophage RNA polymerase in the presence of ribonucleoside triphosphates and a cap dinucleotide such as m(7)Gp(3)G. Unfortunately, because of the presence of a 3'-OH on both the m(7)Guo and Guo moieties, up to half of the mRNAs contain caps incorporated in the reverse orientation. Previously we designed and synthesized two "anti-reverse" cap analogs (ARCAs), m(7)3'dGp(3)G and m(2)(7,3'-)(O)Gp(3)G, that cannot be incorporated in the reverse orientation because of modifications at the C3' position of m(7)Guo. In the present study, we have synthesized seven new cap analogs modified in the C2' and C3' positions of m(7)Guo and in the number of phosphate residues, m(2)(7,2'-)(O)Gp(3)G, m(7)2'dGp(3)G, m(7)2'dGp(4)G, m(2)(7,2'-)(O)Gp(4)G, m(2)(7,3'-)(O)Gp(4)G, m(7)Gp(5)G, and m(2)(7,3'-)(O)Gp(5)G. These were analyzed for conformation in solution, binding affinity to eIF4E, inhibition of in vitro translation, degree of reverse capping during in vitro transcription, capping efficiency, and the ability to stimulate cap-dependent translation in vitro when incorporated into mRNA. The results indicate that modifications at C2', like those at C3', prevent reverse incorporation, that tetra- and pentaphosphate cap analogs bind eIF4E and inhibit translation more strongly than their triphosphate counterparts, and that tetraphosphate ARCAs promote cap-dependent translation more effectively than previous cap analogs.

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Figures

FIGURE 1.
FIGURE 1.
Scheme for organic synthesis of novel “anti-reverse” cap analogs.
FIGURE 2.
FIGURE 2.
Quenching of intrinsic Trp fluorescence in mouse eIF4E (28–217) with m27,3′-OGp3G (Stepinski et al. 2001), m27,3′-OGp4G (compound 14), and m27,3′-OGp5G (compound 18). Binding affinities for cap analogs to eIF4E were calculated as described in Materials and Methods.
FIGURE 3.
FIGURE 3.
Inhibition of globin mRNA translation in rabbit reticulocyte lysate by m7Gp3G (•), m27,2′-OGp4G (compound 15; ▴), and m72′dGp4G (compound 16; ♦). Natural rabbit globin mRNA was translated at 5 μg/mL in a rabbit reticulocyte lysate system, and globin synthesis was detected by incorporation of [3H]Leu into protein as described in Materials and Methods.
FIGURE 4.
FIGURE 4.
Analysis of in vitro synthesized RNAs by enzymatic digestion with either RNase T2 (A,C,E,G) or RNase T2 plus tobacco acid pyrophosphatase (TAP; B,D,F,H) followed by anion-exchange HPLC on a Partisil 10SAX/25 column. Short mRNAs (see Materials and Methods) capped with (A,B) m7Gp3G, (C,D) m27,3′-OGp3G, (E,F) m27,2′-OGp3G (compound 13), and (G,H) m72′dGp3G (compound 12) were generated by in vitro transcription with T7 RNA polymerase in the presence of [α-32P]GTP. Fractions of 1 mL were collected, and the Cerenkov radiation was determined. The elution times of the following standard compounds, detected by UV absorption, are indicated with arrows: 3′-CMP (Cp), 3′-UMP (Up), 3′-AMP (Ap), 3′-GMP (Gp), 3′,5′-m7GDP (pm7Gp), 3′,5′-GDP (pGp), 5′-GDP (ppG), G(5′)p3(5′)G (GpppG), and 5′-GTP (pppG).
FIGURE 5.
FIGURE 5.
Analysis of percent capping. Short mRNAs either (A) uncapped or (B) capped with m27,3′-OGp3G were generated by in vitro transcription with T7 RNA polymerase in the presence of [α-32P]GTP. The RNAs were digested with RNase T2 followed by anion-exchange HPLC as in Figure 4 ▶ except using a steeper phosphate gradient (see Materials and Methods). Fractions of 1 mL were collected, and the Cerenkov radiation was determined. The elution times of the following standard compounds, detected by UV absorption, are indicated with arrows: 5′-GMP (pG), 5′-GDP (ppG), 5′-GTP (pppG), and guanosine-5′-tetraphosphate (ppppG).
FIGURE 6.
FIGURE 6.
Translational efficiency of ARCA-capped mRNAs. Luciferase mRNAs were synthesized in vitro using T7 RNA polymerase and SmaI-digested pluc-A+ in the presence of all four NTPs and Gp3G (▪), m7Gp3G (•), m7Gp4G (first synthesis; ♦), m7Gp4G (second synthesis; ▾), m27,3′-OGp4G (compound 14; ▴), or m27,2′-OGp4G (compound 15; ○), as described in Materials and Methods. The RNAs were translated for 60 min in a rabbit reticulocyte lysate system, and luciferase activity was measured in triplicate in 1-μL aliquots by luminometry; (RLU) relative light units.
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