Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency

Grudzien, Ewa; Stępiński, Janusz; Jankowska-Anyszka, Marzena; Stolarski, Ryszard; Darżynkiewicz, Edward; Rhoads, Robert E.

doi:10.1261/rna.7380904

Cited by 85 publications

(88 citation statements)

References 42 publications

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“…This is in stark contrast to results obtained using an in vitro translation system, in which this cap analogs augments translation compared with m 7 GpppG. 18 This implies that either the Improved RNA vaccines with modified cap analogs AN Kuhn et al methyl-group at the initiating guanosine or the tetraphosphate bridge or both seem to be inhibitory for recruitment of the translation apparatus in DCs.…”

Section: Discussionmentioning

confidence: 74%

“…[18][19][20] Second, these cap analogs contain modifications that have been shown to increase translational efficiency and stability when present at the 5 0 end of mRNAs. 21,22 However, up to now these cap analogs had been primarily studied in cell-free systems for their biochemical characteristics, thus there is scarcity of information regarding their performance in cells.…”

Section: Discussionmentioning

confidence: 99%

“…RNA species linked to phosphorothioate and ARCA cap variants appeared to profit from both a higher functional stability and higher translational efficiency (Table 1). Surprisingly, m 7 Gppppm 7 G-capping of RNA, although reported to augment translation in vitro, 18 resulted in very low protein yields, apparently because of impaired translation and instability of this RNA species in immature DCs.…”

Section: B-s-arca(d1)-capped Rna Is Superior In Ensuring High and Lonmentioning

confidence: 99%

“…For example, 'two-headed' cap analogs of the format m 7 Gp (n) m 7 G, owing to their symmetry, are exclusively incorporated in the correct orientation. 18 Among such symmetric analogs the tetraphosphate m 7 …”

Section: Introductionmentioning

confidence: 99%

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Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

et al. 2010

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Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5 0 mRNA cap analogs on the kinetics of the encoded protein, we found that m 2 7,2 0 ÀO Gpp S pG (b-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as b-S-ARCA(D1)-capped RNA species is superior for protein expression and for efficient priming and expansion of naïve antigen-specific T cells in mice. Our findings establish 5 0 mRNA cap analogs as yet another module for tuning immunopharmacological properties of recombinant antigen-encoding RNA for vaccination purposes.

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Section: Discussionmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Section: B-s-arca(d1)-capped Rna Is Superior In Ensuring High and Lonmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

et al. 2010

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“…Perhaps some other process in 48 S complex formation is rate-limiting. Arguing against this suggestion is the fact that the binding affinity of various cap analogs to eIF4E is positively correlated with the translational efficiency of mRNA capped with those analogs, as measured both in vitro (63,69,83) and in vivo (84). This correlation would not hold if cap-eIF4E interactions did not make a significant contribution to the rate of initiation.…”

Section: Discussionmentioning

confidence: 99%

Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides

Slepenkov

Darżynkiewicz

Rhoads

2006

Journal of Biological Chemistry

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Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5-terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m 7 GpppG, the rate constant of association, k on , was dependent on ionic strength, decreasing progressively up to 350 mM KCl, but the rate constant of dissociation, k off , was independent of ionic strength. Phosphorylation of eIF4E decreased k on by 2.1-2.3-fold at 50 -100 mM KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mM. Contrary to published evidence, eIF4E phosphorylation had no effect on k off . Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single-to double-exponential as the eIF4E concentration was increased. However, measuring k off for dissociation of a pre-formed eIF4E⅐m 7 GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in k off . This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.The efficiency of mRNA translational initiation is strongly enhanced by the 5Ј-terminal cap, m 7 GpppN (1). The cap specifically binds to eIF4E, 2 which may be the first canonical initiation factors to interact with mRNA during its recruitment to the ribosome. eIF4E in turn binds to eIF4G, a protein that also interacts with the RNA helicase eIF4A to promote unwinding of mRNA secondary structure, with the multisubunit factor eIF3 to recruit the 43 S initiation complex, and with the cytoplasmic poly(A)-binding protein to enhance initiation of poly(A)-containing mRNAs. Initiation codon recognition is followed by dissociation of eIFs and joining of the 60 S ribosomal subunit to form the elongation-competent 80 S initiation complex. eIF4E has been extensively investigated in organisms that range from yeast to mammals (2-7). Besides translation, eIF4E also functions in nucleocytoplasmic transport of mRNA, sequestration of mRNA in a nontranslatable state, and stabilization of mRNA against decay in the cytosol (8 -10). The three-dimensional structures of human, mouse, and Saccharomyces cerevisiae eIF4E have been solved (11-13). The complex of full-length human eIF4E with m Cap analogs bind to eIF4E in a tight complex, a step that has been studied primarily by equilibrium techniques (15-33). Intrinsic Trp fluorescence quenching of N-termi...

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Highly Regioselective Methylation of Guanosine Nucleotides: An Efficient Synthesis of 7‐Methylguanosine Nucleotides

Senthilvelan

Shanmugasundaram

Kore

2019

CP Nucleic Acid Chemistry

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This article describes a simple, reliable, efficient, and general method for the synthesis of 7‐methylguanosine nucleotides such as 7‐methylguanosine 5′‐O‐monophosphate (m7GMP), 7‐methylguanosine 5′‐O‐diphosphate (m7GDP), 7‐methyl‐2′‐deoxyguanosine 5′‐O‐triphosphate (m72′dGTP), and 7‐methylguanosine 5′‐O‐triphosphate (m7GTP) starting from the corresponding guanosine nucleotide is described. The present protocol involves methylation reaction of guanosine nucleotide using dimethyl sulfate as a methylating agent and water as a solvent at room temperature to provide the corresponding 7‐methylguanosine nucleotide in good yields with high purity (>99.5%). It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7‐methylguanosine nucleotide. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 7‐methylguanosine nucleotides.

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Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency

Abstract: Gp 3 G, respectively. Relative translational efficiencies could generally be explained in terms of cap affinity for eIF4E, % capping, and % correct orientation. The measurement of all five parameters provides insight into factors that contribute to translational efficiency.

Cited by 85 publications

References 42 publications

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides

Highly Regioselective Methylation of Guanosine Nucleotides: An Efficient Synthesis of 7‐Methylguanosine Nucleotides

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