Locked Nucleic Acid (LNA)-Modified Dinucleotide mRNA Cap Analogue: Synthesis, Enzymatic Incorporation, and Utilization

Abstract

There has been considerable therapeutic interest in the development of human vaccines against cancers and infectious diseases such as HIV and biowarfare agents by using transfected mRNAs for antigenic proteins of interest. The highest expression levels of these proteins are obtained when the transfected mRNA contains 5'-capped ends. In the present study, the locked nucleic acid (LNA)-modified cap analogue 3, m(7(LNA))G[5']ppp[5']G, has been synthesized and its biological properties were examined. The LNA-modified cap analogue was an efficient substrate for T7 RNA polymerase, and the mRNA transcribed, with a poly(A) tail, was efficiently utilized in an in vitro translation process. The RNA with the 5'-LNA-modified cap was found to be approximately 1.61- and 1.28-fold more stable than the RNA with the 5'-standard 4 and ARCA cap, respectively, and approximately 4.23-fold more stable than the uncapped control RNA. The RNA capped with the m(7(LNA))G[5']ppp[5']G 3 cap analogue was translated the most efficiently, with approximately 3.2-fold more activity than the standard cap, m(7)G[5']ppp[5']G 4. Furthermore, we have developed a nonradioactive analytical HPLC assay to determine that the LNA-modified 3 cap analogue was incorporated solely into the forward orientation. Molecular modeling of the m(7(LNA))G[5']ppp[5']G 3 cap analogue with the cap binding protein elF4E complex indicates that the LNA-modified cap-protein complex is more stable by 47.28 kcal/mol as compared to the standard mCAP-protein complex. These findings suggest that the new antireverse cap analogue m(7(LNA))G[5']ppp[5']G 3 is a potential candidate for RNA-based therapeutic vaccine production as well as studying biochemical processes.

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

“…These analogs are modified (or blocked) at the 3′- or 2′-OH hydroxyl of 7-methylguanosine to prevent recognition of this moiety by RNA polymerase [36, 101, 102]. The simplest and most commonly used modification is methylation of one of those groups, but ARCAs containing hydrogen (O–H substitution) or fluorine (O–F substitution) or even bulkier substituents have also been reported [37, 38, 103–106]. Importantly, chemical modifications at these positions do not affect interactions with eIF4E or translation efficiency of such capped mRNAs, although, in the case of some bulkier substituents, the translation efficiency can be slightly decreased in comparison to ARCAs carrying simply a methyl group [37, 38].…”

Design and Facile Synthesis of New Dinucleotide Cap Analog Containing Both 2′ and 3′-OH Modification on M7Guanosine Moiety

“…Subsequently, it has been reported that 2 -OH modification on m 7 G moiety displays as an ARCA property. [12] Following these findings, several other ARCA-type modifications, such as 2 -fluoro, [13] 2 -araF, [14] LNA-m 7 G, [15] 2 -allyl, [16] and 2 ,3 -O,O-isopropylidene, [17,18] have been reported. It is noteworthy that these ARCA analogs outweighs standard cap analog in terms of their translational properties.…”

“…We envisaged that the introduction of acetyl group on both 2 and 3 -OH groups on m 7 G moiety would improve translational properties as well as intracellular stability due to the presence of bulky groups. Our continuous interest in the design of new cap analogs to improve the translational efficiency of capped mRNA, [13][14][15][16][17] prompted us to synthesize diacetyl cap analog and study its biological applications. Herein, we report the first example for the synthesis of cap analog containing 2 ,3 diacetyl group on m 7 G moiety such as m 7,2 ,3 -diacetyl G [5 ]ppp [5 ]G.…”

Highly RegioselectiveC-5 Iodination of Pyrimidine Nucleotides and Subsequent Chemoselective Sonogashira Coupling with Propargylamine

“…Given the ever growing molecular biology applications, the development of a new and efficient gram-scale method for the preparation of aminopropargyl nucelotides with high purities would be highly valuable to meet the application of nucleic acid chemistry demand. Our continuous interest in the area of nucleic acid chemistry [20][21][22][23][24][25] prompted us to explore the possibility of palladium-catalyzed Sonogashira coupling reaction of 5-iodo-pyrimidine-5 -triphosphates with propargylamine for the synthesis of 5-aminopropargyl-pyrimidine-5 -triphosphates. In our preliminary communications, we reported the first example of highly regioselective iodination of uridine nucleotide [26] and also the first example of a palladiumcatalyzed Sonogashira coupling of 5-iodouridine triphosphates with propargylamine, leading to the formation of 5-aminopropargyl-uridine triphosphates in good yields with high purities.…”