The basic problem in the application of catalytic ribonucleic acids in vivo is their dependence on relatively high concentration of magnesium ions. At experimental, in vitro conditions, the concentration of Mg²⁺ is usually in the range of 10 mM, which is much higher than the concentration within the cell. In recent years, there were several reports which questioned the general belief that magnesium plays a catalytic role in ribozyme-dependent reactions. It is now becoming more and more clear that the role of Mg²⁺ is rather structural.

One of the methods used to increase the catalytic activity of hammerhead ribozymes at low magnesium concentration is the introduction of additional tertiary interactions which stabilize an active conformation of the ribozymes' catalytic center. We designed an AUG-targeted hammerhead ribozyme against a sequence encoding gp41 protein from HIV-1. The stabilizing element of the new ribozyme consists of a tetraloop and a tetraloop receptor (TL:TLR) localized within the catalytic core and and additional domain at the 5' end of the construct, respectively. The analysis of the catalytic activity of he TL:TLR-containing ribzozyme with the classical hammerhead and DNAzyme targeting the same region of the gp41 mRNA revealed that it is more efficient in the whole spectrum of magnesium concentrations. At low levels of Mg²⁺ (below 1 mM) TL:TLR-stabilized ribozyme shows approximately two-fold higher activity than other catalytic nucleic acids used in the study. The efficiency of the new construct was also confirmed in cultured cells in the experiments with the inhibition of expression of the chimeric GFP gene.

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