The sequence specificity of hammerhead ribozyme cleavage has been re-evaluated with respect to the NUH rule. Contrary to previous reports it was found that substrates with GAC triplets were also cleaved. This was established in three different sequence contexts. The rate of cleavage under single turnover conditions was between 3 and 7% that of cleavage 3' of GUC. Specificity of cleavage of substrates containing a central A in the cleavable triplet can be described as NAH, where N can be any nucleotide and H any nucleotide but G. As cleavage 3' of NCH triplets has recently been described, the NUH rule can be reformulated to NHH.

Developments in the hammerhead ribozyme field during the last two years are reviewed here. New results on the specificity of this ribozyme, the mechanism of its action and on the question of metal ion involvement in the cleavage reaction are discussed. To demonstrate the potential of ribozyme technology examples of the application of this ribozyme for the inhibition of gene expression in cell culture, in animals, as well as in plant models are presented. Particular emphasis is given to critical steps in the approach, including RNA site selection, delivery, vector development and cassette construction.

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.

The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chemically modified mRNA cap analogs contain a unique cap structure, m 7 G[5′]ppp[5′]N (where N=G, A, C or U), present at the 5′‐end of many eukaryotic cellular and viral RNAs and several non‐coding RNAs. The chemical modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide containing a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog containing N1 modification, cap analog containing N2 modification, dinucleotide containing fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addition, the biological applications of these novel cap analogs are delineated.