The synthesis of isopropylidene mRNA cap analogs modified with phosphorothioate moiety and their evaluation as promoters of mRNA translation (original) (raw)

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

RNA, 2004

Synthetic analogs of the N7-methylated guanosine triphosphate cap at the 5 end of eukaryotic mRNAs and snRNAs have played an important role in understanding their splicing, intracellular transport, translation, and turnover. We report here a new series of N7-benzylated dinucleoside tetraphosphate analogs, b 7 Gp 4 G, b 7 m 3-O Gp 4 G, and b 7 m 2 Gp 4 G, that extend our knowledge of the role of the cap in translation. We used these novel analogs, along with 10 previously synthesized analogs, to explore five parameters: binding affinity to eIF4E, inhibition of cap-dependent translation in a rabbit reticulocyte lysate system, efficiency of incorporation into RNAs during in vitro transcription (% capping), orientation of the analog in the synthetic mRNA (% correct orientation), and in vitro translational efficiency of mRNAs capped with the analog. The 13 cap analogs differed in modifications of the first (distal) and second (proximal) guanine moieties, the first and second ribose moieties, and the number of phosphate residues. Among these were analogs of the naturally occurring cap m 3 2,2,7 Gp 3 G. These compounds varied by 61-fold in affinity for eIF4E, 146-fold in inhibition of cap-dependent translation, 1.4-fold in % capping, and 5.6-fold in % correct orientation. The most stimulatory analog enhanced translation 44-fold compared with uncapped RNA. mRNAs capped with b 7 m 2 Gp 4 G, m 7 Gp 3 m 7 G, b 7 m 3-O Gp 4 G, and m 7 Gp 4 m 7 G were translated 2.5-, 2.6-, 2.8-, and 3.1-fold more efficiently than mRNAs capped with m 7 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.

Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells

RNA, 2007

Capped RNAs synthesized by in vitro transcription have found wide utility for studying mRNA function and metabolism and for producing proteins of interest. We characterize here a recently synthesized series of cap analogs with improved properties that contain a sulfur substitution for a nonbridging oxygen in either the a-, b-, or g-phosphate moieties, m 2 7,29-O Gppp S G, m 2 7,29-O Gpp S pG, and m 2 7,29-O Gp S ppG, respectively. The new compounds were also modified at the 29-O position of the m 7 Guo to make them anti-reverse cap analogs (ARCAs), i.e., they are incorporated exclusively in the correct orientation during in vitro transcription. Each of the S-ARCAs exists in two diastereoisomeric forms (D1 and D2) that can be resolved by reverse-phase HPLC. A major in vivo pathway for mRNA degradation is initiated by removal of the cap by the pyrophosphatase Dcp1/Dcp2, which cleaves between the aand b-phosphates. Oligonucleotides capped with m 2 7,29-O Gpp S pG (D2) were completely resistant to hydrolysis by recombinant human Dcp2 in vitro, whereas those capped with m 2 7,29-O Gpp S pG (D1) and both isomers of m 2 7,29-O Gppp S G were partially resistant. Luciferase mRNA capped with m 2 7,29-O Gpp S pG (D2) had a t 1/2 of 257 min in cultured HC11 mammary epithelial cells compared with 86 min for m 7 Gp 3 G-capped mRNA. Luciferase mRNAs capped with m 2 7,29-O Gpp S pG (D1) and m 2 7,29-O Gpp S pG (D2) were translated 2.8-fold and 5.1-fold, respectively, more efficiently in HC11 cells than those capped with m 7 Gp 3 G. The greater yield of protein due to combining higher translational efficiency with longer t 1/2 of mRNA should benefit applications that utilize RNA transfection such as protein production, anti-cancer immunization, and gene therapy.

Synthesis and properties of mRNA cap analogs containing imidodiphosphate moiety—fairly mimicking natural cap structure, yet resistant to enzymatic hydrolysis

Bioorganic & Medicinal Chemistry, 2012

We describe synthesis and properties of eight dinucleotide mRNA 5 0 cap analogs containing imidodiphosphate moiety within 5 0 ,5 0 -tri-or tetraphosphate bridge (NH-analogs). The compounds were obtained by coupling an appropriate nucleoside 5 0 -imidodiphosphate with nucleotide P-imidazolide mediated by divalent metal chloride in anhydrous DMF. To evaluate the novel compounds as tools for studying capdependent processes, we determined their binding affinities for eukaryotic translation initiation factor 4E, susceptibilities to decapping pyrophosphatase DcpS and, for non-hydrolysable analogs, binding affinities to this enzyme. The results indicate that the O to NH substitution in selected positions of oligophosphate bridge ensures resistance to enzymatic decapping and suggest that interactions of NH-analogs with cap binding proteins fairly mimic interactions of unmodified parent compounds. Finally, we identified NH-analogs as potent inhibitors of cap-dependent translation in cell free system, and evaluated their utility as reagents for obtaining 5 0 capped mRNAs in vitro to be rather moderate.

SYNTHESIS AND PROPERTIES OF mRNA CAP ANALOGS CONTAINING PHOSPHOROTHIOATE MOIETY IN 5′,5′-TRIPHOSPHATE CHAIN

Nucleosides, Nucleotides and Nucleic Acids, 2005

We describe synthesis and properties of eight dinucleotide mRNA 5 0 cap analogs containing imidodiphosphate moiety within 5 0 ,5 0 -tri-or tetraphosphate bridge (NH-analogs). The compounds were obtained by coupling an appropriate nucleoside 5 0 -imidodiphosphate with nucleotide P-imidazolide mediated by divalent metal chloride in anhydrous DMF. To evaluate the novel compounds as tools for studying capdependent processes, we determined their binding affinities for eukaryotic translation initiation factor 4E, susceptibilities to decapping pyrophosphatase DcpS and, for non-hydrolysable analogs, binding affinities to this enzyme. The results indicate that the O to NH substitution in selected positions of oligophosphate bridge ensures resistance to enzymatic decapping and suggest that interactions of NH-analogs with cap binding proteins fairly mimic interactions of unmodified parent compounds. Finally, we identified NH-analogs as potent inhibitors of cap-dependent translation in cell free system, and evaluated their utility as reagents for obtaining 5 0 capped mRNAs in vitro to be rather moderate.

Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppG

RNA (New York, N.Y.), 2001

The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has been of considerable value in a variety of applications. However, Pasquinelli et al. [RNA (1995) 1:957-967] found that one-third to one-half of the caps are incorporated in the reverse orientation, that is, with the m7G moiety of m7GpppG linked by a 3'-5' phosphodiester bond to the first nucleotide residue of the RNA chain. Such reverse caps are unlikely to be recognized by eIF4E, based on previous studies, and thus complicate any comparison of the translational efficiencies of in vitro-synthesized mRNAs. We therefore designed two novel cap analogs, P(1)-3'-deoxy-7-methyguanosine-5' P3-guanosine-5' triphosphate and P(1)-3'-O,7-dimethylguanosine-5' P3-guanosine-5' triphosphate, that are, theoretically, incapable of being incorporated in the reverse orientation. The key reactions of pyrophosphate bond formation were achieved in anhydrous dimethylformamide solut...

Synthesis of the first double-functionalized dinucleotide mRNA cap analogue for its specific labeling

Tetrahedron Letters, 2017

The modification of various important nucleotide-based molecules (such as nucleotides, RNA, DNA, oligonucleotides) with fluorophores, affinity tags and reactive moieties is of enormous utility for studying their localization, structure and dynamics, as well as diverse biological functions involving their interacting partners. Herein, we report chemical methodology in which the dinucleotide mRNA cap analogue is doubly modified within its second nucleotide. The prepared dinucleotide contains an alkyne at the N2-position of guanine, and levulinic acid within the ribose moiety. Such modifications may be further used for specific labeling of the cap, for instance with a fluorophore that will allow the molecule to be tracked inside the cell and an attachment cell-penetrating peptide that will help to deliver it to the area of interest. Exemplar molecules were attached in order to demonstrate the utility of the newly synthesized cap analogue.

mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation

Nucleic acids research, 2017

Analogues of the mRNA 5΄-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e. one of the bridging O atom substituted with CCl2 or CF2) and their properties in the context of cellular translational and decapping machineries, compared to phosphate-unmodified and previously reported CH2-substituted caps. The analogues were bound tightly to eukaryotic translation initiation factor 4E (eIF4E), with CCl2-substituted analogues having the highest affinity. When incorporated into mRNA, the CCl2-substituted dinucleotide most efficiently promoted cap-dependent translation. Moreover, the CCl2-analogues were potent inhibitors of translation in rabbit reticulocyte lysate. The crystal structure of eIF4E in complex with the CCl2-analogue revealed a significantly di...

N2 modified dinucleotide cap analogs as a potent tool for mRNA engineering

RNA

mRNA-based vaccines are relatively new technologies that have been in the field of interest of research centers and pharmaceutical companies in recent years. Such therapeutics are an attractive alternative for DNA-based vaccines since they provide material that can be used with no risk of genomic integration. Additionally, mRNA can be quite easily engineered to introduce modifications for different applications or to modulate its properties, for example, to increase translational efficiency or stability, which is not available for DNA vectors. Here, we describe the use of N2 modified dinucleotide cap analogs as components of mRNA transcripts. The compounds obtained showed very promising biological properties while incorporated into mRNA. The presented N2-guanine modifications within the cap structure ensure proper attachment of the dinucleotide to the transcripts in the IVT reaction, guarantees their incorporation only in the correct orientation, and enables highly efficient transla...

Synthesis of Novel mRNA 5′ Cap-Analogues: Dinucleoside P, P-Tri-, P, P-Tetra-, and P, P-Pentaphosphates

Nucleos Nucleot Nucleic Acids, 2003

A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying the mechanism of protein translation. Dinucleoside P 1 , P 3-triP P 1 , P 4-tetra-and P 1 , P 5-pentaphosphates, linked by a 5 0-to-5 0 phosphate bridge and composed of modified 7-methylguanosine and guanosine, have been synthesized. The hydroxyl group (2 0 OH or 3 0 OH) in 7-metylguanosine moiety was replaced by-OCH 3 or-H in order to obtain the cap analogues capable to be correctly incorporated into synthetic mRNA transcripts. Tri-, tetra-, and pentaphosphates were prepared by ZnCl 2 catalyzed condensation in DMF of derivatives of the 7-methylguanosine diphosphates with the guanosine mono-, di-and triphosphate P-imidazolides, respectively. The structures of the novel compounds were established by means of 1 H and 31 P NMR spectra.

Synthesis of Anti-Reverse Cap Analogs (ARCAs) and their Applications in mRNA Translation and Stability

2007

Synthetic capped RNA transcripts produced by in vitro transcription in the presence of m 7 Gp 3 G have found a wide application in studying such processes as mRNA translation, pre-mRNA splicing, mRNA turnover, and intracellular transport of mRNA and snRNA. However, because of the presence of a 3 0 -OH on both m 7 Guo and Guo moieties of the cap structure, one-third to one-half of the mRNAs contain a cap incorporated in the reverse orientation. The reverse cap structures bind poorly to eIF4E, the cap binding protein, and reduce overall translational efficiency. We therefore replaced the conventional m 7 Gp 3 G cap by ''anti-reverse'' cap analogs (ARCAs) in which the 3 0 -OH of m 7 Guo moiety was substituted by 3 0 -deoxy or 3 0 -O-methyl groups, leading to m 7 3 0 dGp 3 G or m 2 7,3 0 -O Gp 3 G, respectively. The class of ARCAs was extended to analogs possessing an O-methyl group or deoxy group at C2 0 of m 7 Guo. We have also developed a series of ARCAs containing tetra-and pentaphosphates. mRNAs capped with various ARCAs were translated 1.1-to 2.6-fold more efficiently than their counterparts capped with m 7 Gp 3 G in both in vitro and in vivo systems. In a separate series, a methylene group was introduced between the aand b-, or band g-phosphate moieties, leading to m 2 7,3 0 -O Gpp CH2 pG and m 2 7,3 0 -O Gp CH2 ppG. These analogs are resistant to cleavage by the decapping enzymes Dcp1/Dcp2 and DcpS, respectively. mRNA transcripts capped with m 2 7,3 0 -O Gpp CH2 pG were more stable when introduced into cultured mammalian cells. In this chapter, we describe the synthesis of representative ARCAs and their biophysical and biochemical characterization, with emphasis on practical applications in mRNA translation.