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.