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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.

Synthesis of the nucleoside building block of the 6-keto derivative of 2'-deoxy-5-methylcytidine (m5oxC) as an analog of an N3-protonated cytosine derivative is described. A series of 15mer oligonucleotides containing either four or six m5oxC residues has been prepared by chemical synthesis. Complexation of the 15 residue oligonucleotides with target 25mer duplexes results in DNA triplexes containing T-A-T and m5oxC-G-C base triplets. When the m5oxC-G-C base triplets are present in sequence positions that alternate with TAT base triplets, DNA triplexes are formed with Tm values that are pH independent in the range 6.4-8.5. A 25mer DNA duplex containing a series of five contiguous G-C base pairs cannot be effectively targeted with either m5C or M5oxC in the third strand. In the former case charge-charge repulsion effects likely lead to destabilization of the complex, while in the latter case ineffective base stacking may be to blame. However, if the m5C and M5oxC residues are present in the third strand in alternate sequence positions, then DNA triplexes can be formed with contiguous G-C targets even at pH 8.0.

No abstract

A chiral acyclic nucleoside, one in which the ribose carbohydrate has been replaced with a glycerol-based linker, is prepared by glycosylating guanine at the N7-nitrogen. The stereochemically pure derivative is converted to a DMT-protected phosphoramidite for incorporation into DNA sequences. Sequence containing the acyclic N7-dG nucleoside are capable of forming DNA triplexes in which it is likely that the N1-H and N2-amino groups of the N7-dG are involved in recognition of the guanine base in G-C base pairs.