Planar perylene-and naphthalene-based diimide linkers can be employed to tether the Watson-Crick and the Hoogsteen strands of a DNA triplex, thus providing conjugates capable of targeting singlestranded nucleic acids with the formation of hairpin triplexes. The planar linkers are designed to bridge the terminal base triplet of the three-stranded complex and provide base-stacking interactions with all three residues. Sixteen complexes have been prepared, eight with each linker, half with RNA (R) targets and half with DNA (D) targets. The conjugate sequences are composed of two strands of DNA, two of 2′-O-methyl RNA (M), or one of each. In comparison to similar complexes formed with a hexa(ethylene glycol) linker, the planar linkers enhance the T M values for the complexes by as much as 28 °C with ∆G values indicating as much as 12.3 kcal/mol of stabilization relative to the simple glycol linker. All sixteen complexes have been characterized by T M measurements and ∆G determinations. That π-stacking interactions are present between the linkers, and the nucleobases can be inferred from the quenching of the perylene fluorescence upon complex formation, and the observation of an absorbance vs temperature transition for the naphthalene-based linker at 383 nm and for the perylene-based linker monitored at 537 nm.

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Five modified hammerhead ribozyme/substrate complexes have been prepared in which individual adenosine N3-nitrogens have been excised and replaced with carbon. The modified complexes were chemically synthesized with the substitution of a single 3-deazzaadenosine (c3A) base analogue for residues A6, A9, A13, A14, or A15.1. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/K(M), for the c3A6, c3A9, and c3A14 complexes were only marginally reduced (< or = 5-fold) relative to the native complex. By comparison, the cleavage efficiencies for the c3A13 and c315.1 complexes were reduced by 9-fold and 55-fold, respectively. these reductions in cleavage efficiency are primarily a result of lower kcat values. Profiles of pH and cleavage rate suggest that the chemical cleavage step is the rate-limiting reaction for these complexes. These results suggest that the N3-nitrogen of the A13 residue and particularly the A15.1 residue in the hammerhead ribozyme/substrate complex are critical for transition state stabilization and efficient cleavage activity. We have additionally compared the locations of these critical functional groups, as well as those identified from other studies, with recent crystallographic analyses. In some cases, the critical functional groups are clustered around proposed metal binding sites and may reflect functional groups critical for binding the metal cofactor. In other cases, clusters of functional groups may form a network of hydrogen bonds necessary for transition state stabilization.

Three modified hammerhead ribozyme/substrate complexes have been prepared in which individual uridine O2-carbonyls have been eliminated. The modified complexes were chemically synthesized with the substitution of a single 2-pyridone (2P) base analogue for residues U4, U7, and U16.1. Steady-state kinetic analyses indicate that the cleavage efficiencies for the U7 and U16.1 complexes were not significantly reduced relative to the native complex as measured by kcat/KM. The cleavage efficiency for the 2P4 complex, with the analogue present within the uridine loop, was reduced by greater than 2 orders of magnitude. This significant reduction in catalytic efficiency was due primarily to a decrease in kcat. The pH vs cleavage rate profile suggests that the O2-carbonyl of the U4 residue of the hammerhead complex is critical for transition state stabilization and efficient cleavage activity. The results of a Mg2+ rescue assay do not implicate the O2-carbonyl of U4 in an interaction with a divalent metal ion. In addition, the results of a ribozyme folding assay suggest that the presence of the 2P4 within the uridine loop does not alter the folding pathway (relative to the native sequence) both in the absence and in the presence of Mg2+. The O2-carbonyl of U4 appears oriented toward the interior of the catalytic pocket where it may be involved in a critical hydrogen bonding interaction necessary for transition state stabilization.