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

2007; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(07)31011-2

1557-7988

Ewa Grudzien‐Nogalska, Janusz Stȩpiński, Jacek Jemielity, Joanna Zuberek, Ryszard Stolarski, Robert E. Rhoads, Edward Darżynkiewicz,

RNA modifications and cancer

Synthetic capped RNA transcripts produced by in vitro transcription in the presence of m7Gp3G 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′‐OH on both m7Guo 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 m7Gp3G cap by “anti‐reverse” cap analogs (ARCAs) in which the 3′‐OH of m7Guo moiety was substituted by 3′‐deoxy or 3′‐O‐methyl groups, leading to m73′dGp3G or m27,3′‐O Gp3G, respectively. The class of ARCAs was extended to analogs possessing an O‐methyl group or deoxy group at C2′ of m7Guo. 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 m7Gp3G in both in vitro and in vivo systems. In a separate series, a methylene group was introduced between the α‐ and β‐, or β‐ and γ‐phosphate moieties, leading to m27,3′‐OGppCH2pG and m27,3′‐OGpCH2ppG. These analogs are resistant to cleavage by the decapping enzymes Dcp1/Dcp2 and DcpS, respectively. mRNA transcripts capped with m27,3′‐OGppCH2pG 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.