RESUMO
An efficient synthesis of new cap analogs containing 7-deazaguanosine moiety such as m(7)G[5']ppp[5'](7-deaza)G and m2(7,3'O)G[5']ppp[5'](7-deaza)G is described. The biological substrate validation of these new cap analogs is evaluated with respect to its capping efficiency and in vitro T7 RNA polymerase transcription using standard cap m7G[5']ppp[5']G as a control. The capping efficiency and HPLC data reveal that these new analogs are not the substrate for T7 RNA polymerase or SP6 RNA polymerase. The present study highlights the importance of the presence of nitrogen atom at N7-position of the guanosine moiety for the polymerase recognition.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Guanosina/análogos & derivados , Proteínas de Ligação ao Cap de RNA/química , Proteínas de Ligação ao Cap de RNA/síntese química , Transcrição Gênica/fisiologia , Cromatografia Líquida de Alta Pressão , Guanosina/química , Técnicas In Vitro , Estrutura MolecularRESUMO
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.
Assuntos
Oligonucleotídeos/química , Análogos de Capuz de RNA/síntese química , RNA Polimerases Dirigidas por DNA/metabolismo , Fosfatos de Dinucleosídeos , Terapia Genética , Humanos , Oligonucleotídeos/metabolismo , Biossíntese de Proteínas , Análogos de Capuz de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/biossíntese , Transfecção , Vacinas/genética , Proteínas Virais/metabolismoRESUMO
Design, synthesis, and biological evaluation of 2'-fluoro-substituted cap analogs, i.e., m(7,2'F)G[5']ppp[5']G and m(7,2'F)G[5']ppp[5']m(7)G are described. Structures were confirmed by (1)H, (31)P, (19)F NMR and MS data. The effects of the 2'-fluoro-substituted moiety from the normal and N(7) double methylated mCAP were evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured HeLa cells. Luciferase fusion protein production was monitored by measuring the luciferase activity. mRNA poly(A) capped with 2'-fluoro-substituted cap analogs, (m(7,2'F)G[5']ppp[5']G) and (m(7,2'F)G[5']ppp[5']m(7)G), were translated approximately 2.4- and 2.5-fold more efficiently, respectively, than mRNA capped with conventional m(7)G[5']ppp[5']G.
