Recent Developments in 5‘-Terminal Cap Analogs: Synthesis and Biological Ramifications.

Anilkumar   R.   Kore; Irudaya      Charles; Muthian      Shanmugasundaram; Zejun      Xiao; Richard   C.   Conrad

Abstract

The 5‘-terminus of eukaryotic messenger RNA (mRNA) molecules contains a unique ‘cap’ structure: 7- methylguanosine (m7G) linked by a 5‘-5‘-triphosphate bridge to the first nucleoside in the standard polymer chain. This 5‘- cap is recognized by numerous enzymes involved in the transport and translation of mRNA, as well as its processing, both in terms of generating mature mRNA from the initial transcript and its natural degradation. In vitro generation of capped RNAs uses a cap analog in which two nucleosides are joined in a 5‘-5‘ triphosphate linkage, allowing incorporation of the cap along with the first nucleotide in the polymer chain. The conventional cap analog, m7G[5‘]ppp[5‘]G, has a 3‘-OH on both nucleoside moieties, resulting in the incorporation of the dinucleotide in either orientation. This results in one-third to one-half of the mRNAs containing a non-methylated cap and a methylated initial nucleotide. These ‘reverse cap’ structures bind poorly to elF4E and other cap binding proteins, greatly reducing the overall translational efficiency in vitro and biological activity in vivo. This drawback of the conventional cap analog has been addressed by creating an “anti-reverse cap analog” (ARCA), m2 7, 3-OG[5‘]ppp[5‘]G, in which the 3‘-OH of the ribose connected to the m7Guo moiety is replaced with a 3‘-O-methyl group, allowing incorporation only in the forward orientation due to the presence of the single extendable 3‘-OH group. In addition to the chemical modification at the 3‘-OH group of the ribose connected to the m7G moiety, modifications at the 2‘-OH group also preclude attachment of a second nucleotide by polymerase, again forcing the forward orientation. This mini-review focuses on recent developments in the synthesis of novel cap analogs, highlighting modifications at several positions including; the heterocyclic bases, the sugars, and the α-, β-, and γ-phosphates in the triphosphate chain, and assessing their impact on biological activity.

Keywords: Cap analogs, capping efficiency, in vitro transcription, translation efficiency, luciferase activity, HeLa cells, antireverse cap analog (ARCA), elF4E, cap analog inhibition, mammalian cells