锁核酸
信使核糖核酸
化学
核酸
核糖核酸
分子生物学
T7 RNA聚合酶
转染
酶
聚合酶
生物化学
生物
基因
大肠杆菌
噬菌体
作者
Anilkumar R. Kore,Muthian Shanmugasundaram,Irudaya Charles,Alexander V. Vlassov,Timothy Barta
摘要
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, m7(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 ∼1.61- and 1.28-fold more stable than the RNA with the 5′-standard 4 and ARCA cap, respectively, and ∼4.23-fold more stable than the uncapped control RNA. The RNA capped with the m7(LNA)G[5′]ppp[5′]G 3 cap analogue was translated the most efficiently, with ∼3.2-fold more activity than the standard cap, m7G[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 m7(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 m7(LNA)G[5′]ppp[5′]G 3 is a potential candidate for RNA-based therapeutic vaccine production as well as studying biochemical processes.
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