Artificial Ribonucleases

Incubation conditions: 0.5 mM 3'-[32P]-tRNAPhe, 50 mM Tris-HCl (pH 7.0), 200 mM KCl, 1 mM EDTA, 37 C.

Cartoons and data for an oligonucleotide containing 24 imidazole residues (conjugate), synthesized using MOX precursor amidites, behaving as an artificial ribonuclease. The core 16-mer deoxyoligonucleotide, 5'-ATC GAA CAC AGG ACC T-3', that is a complement to the loop region of tRNAPhe, was built up with modifying precursors and a branching unit.  The prepared multiple MOX precursors were then functionalized with histamine and deprotected in the usual way. The gel shows the disappearance of the target tRNA and the appearance of two shorter bands (two cleavage sites) at the 10 mM range of the conjugate. The kinetics of site-directed tRNAPhe cleavage is shown graphically for the 0.5 mM to 100 mM ranges of this artificial ribonuclease. This data suggests that a fast cleavage (30 min) can be achieved using 50 mM of conjugate (100-fold excess over target RNA).

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