The goal of this project is the development of molecules that cleave RNA with a high degree of sequence-specificity and show catalytic turnover in the cleavage of RNA (artificial restriction enzymes). Artificial restriction enzymes will be constructed from metal transesterification catalyst and an oligodeoxynucleotide that is complementary in sequence to an RNA. The unique feature of this proposal is the use of metal transesterification catalysts to cleave RNA. Transesterification of RNA is an example of a substitution reaction at phosphorus(V) which is catalyzed by metal ions; in this reaction, a strand of RNA is neatly cleaved by breaking a single phosphorus-oxygen bond. Because of metal transesterification catalysts are specific for RNA and do not cleave DNA, do not produce diffusible radicals to cleave RNA and show catalytic turnover, artificial enzymes constructed from these catalysts will have many desirable properties. N-ethyl-ethylenediamine-N, N'-diacetate and iminodiacetate will be attached to oligodeoxynucleotides. Tether length and metal ion (lead(II), lanthanum(III) or zinc(II) will be varied in order to optimize specific cleavage. Experimentation with oligonucleotide length and the temperature of the cleavage reaction will enable us to determine conditions where catalytic turnover may occur. The sequence-specific cleavage of t-RNAphe with artificial restriction enzymes will be examined, and the site of cleavage will be determined by standard protocol for nucleic acid sequencing. In addition, we propose to further develop a new class of catalysts based on zinc(II), magnesium(II) or nickel(II) complexes of macrocyclic polyamines containing a pendant pyridine. The metal as a Lewis acid may work in concert with the pendant pyridine to catalyze transesterification by a bifunctional mechanism. To accelerate catalytic RNA cleavage, the position of the pyridine with respect to the metal will be changed by varying the position of attachment of the pyridine and the length of the linker arm. In the long term, these artificial restriction enzymes will be developed for use as selective inhibitors of gene expression and as reagents for laboratory manipulations of RNA.
