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Electron paramagnetic resonance (EPR) spectroscopy is used to examine changes in internal structure and dynamics of the hammerhead ribozyme upon metal ion induced folding, changes in pH, and the presence and absence of ribozyme inhibitors. Results reveal that different U7 dynamics are observed in the presence of catalytically efficient divalent metal ion concentrations than in the presence of high concentrations of monovalent metal ions.

The metal ion specificity of the hammerhead (HH) ribozyme reaction upon sulfur substitution of the 2'-nucleophile is examine to characterize the behavior of 2'-mercapto oligonucleotides in biochemical reactions. The mechanistic evidence reveals that mercaptonucleotides can be used to generate carbon radical site specifically within structures RNA molecules, thereby providing possible probes for cross-linking and other biophysical studies.

Research has been conducted on the hammerhead ribozyme with a phosporodithioate linkage. The role of the metal ion in the catalysis has been investigated.

An investigation is presented into the coordination environment of a hammerhead ribozyme using EPR and continuous-wave Q-band (34 GHz) electron double-resonance spectroscopies.

The mechanism of conformation and folding of the four-way RNA junction of the hairpin ribozyme was investigated using fluorescence resonance energy transfer. It was shown that the junction exhibits a square configuration of coaxially stacked arms based on A on D and B on C stacking in the absence of added metal ions. The junction undergoes an ion-induced transition to an antiparallel conformation with the addition of magnesium ions. These results are in agreement with folding induced by the binding of a single ion.

A study was conducted to examine the influence of metal ions on the activity and global structure of hairpin ribozymes in their natural junction conformation. T7 RNA polymerase was utilized to determine RNA from synthetic DNA templates while phosphoramidite chemistry was carried out to synthesize DNA and mixed DNA-RNA oligonucleotides. Results indicated that natural ribozyme activity can be influenced by the conformation of the four-way junction.

The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple [Mg.sup.2+] ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in [Ca.sup.2+] were enriched over 18 generations of selection for catalysis in the presence of [Ca.sup.2+], which is normally disfavored relative to [Mg.sup.2+]. Although a wide spectrum of mutations was found in the generation-18 clones, only a single point mutation was common to all clones: a cytosineto-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of [Ca.sup.2+] selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in [Mg.sup.2+] binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4J2/4 domain forms a portion of the ribozyme's active site.

In a previous examination using natural all-RNA substrates that contained either a 5[prime]-oxy or 5[prime]-thio leaving group at the cleavage site, we demonstrated that (i) the attack by the 2[prime]-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5[prime]-thio group (R11S) and (ii) the departure of the 5[prime] leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy diagrams for these reactions were provided in our previous publication. In this report we found that the rate of cleavage of R11O by a hammerhead ribozyme was enhanced 14.fold when [Mg.sup.2+] ions were replaced by [Mn.sup.2+] ions, whereas the rate of cleavage of R11S was enhanced only 2.2-fold when [Mg.sup.2+] ions were replaced by [Mn.sup.2+] ions. This result appears to be exactly the opposite of that predicted from the direct coordination of the metal ion with the leaving 5[prime]-oxygen, because a switch in metal ion specificity was not observed with the 5[prime]-thio substrate. However, our quantitative analyses based on the previously provided energy diagram indicate that this result is in accord with the double-metal-ion mechanism of catalysis.

Theoretical studies using ab initio calculations and kinetic experiments demonstrate the ability of magnesium ion to cleave a phosphorus-oxygen bond by directly coordinating with the translating oxygen. This direct coordination favors the second step of the hammerhead ribozyme reactions. A comparative study of the stabilities of oxyphosphoranes with different ionic valences reveals that stability is increased by the protonation of a dianionic species.

The divalent metal ions Pb2+ and Mg2+ can cleave a catalytic RNA molecule made up of an asymmetric internal loop of six to 10 nucleotides. This catalytic RNA is in the form of a small metalloribozyme with a two-step reaction mechanism; first a 2',3'-cyclic phosphodiester produces a 3' phosphomonoester which then terminates with a 5' cleavage product. This two-step process is similar to what occurs in many protein ribonucleases but has not hitherto been detected in catalytic RNA reactions.

An examination of activity of the two ribozymes from hepatitis delta virus in monovalent salts was carried to compare the activity in [Mg.sup.2+]. The findings support the idea that monovalent cations could partially substitute for divalent metal ions in the HDV ribozymes, although a divalent metal ion is more effective in supporting catalysis.

Metal ions facilitate the folding of the hairpin ribozyme but do not participate directly in catalysis. A model of the [Co(NH3)6](super 3+)-mediated catalytic core generated by MC-SYM that is consistent with these data.

The adenosine derivative of deoxyadenosine by an amide linker (X) incorporated in a DNA stand promoted the site-selective hydrolysis of the complementary RNA in the presence of MgCl(sub 2). Natural ribozymes catalyzes the site-selective RNA cleavage reaction by utilizing metal ions and nucleotide bases as catalysts, in which the nucleotide subject to be cleaved generally is located in an unpaired region involving the tertiary interactions.

The function of metal ions in ribozyme catalysis was studied by examining the role of hammerhead ribozymes. The link between metal ions and pro-R oxygen was investigated using spectroscopy. Single turnover activity assays were performed to examine the contribution of Hg(II)-binding to ribozyme activity. Results showed evidence of the relationship between the inner-sphere coordination of Hg(II) and the nonbridging sulfur at the cleavage site of the hammerhead derivative.

The reverse branching in the lariat intron-3-exon intermediate has been investigated. In this reaction, the excised lariat intron and the substrate 5'-exon can be preincubated and folded separately to allow the measurement of the catalytic rate. The results showed that substitution of magnesium ions by manganese ions enhanced the rate of the first transesterification reaction by two orders of magnitude but had effect on the second transesterification reaction or the 5' splice site cleavage by hydrolysis.