THE EMBEDDED RIBONUCLEOTIDE ASSAY - A CHIMERIC SUBSTRATE FOR STUDYINGCLEAVAGE OF RNA BY TRANSESTERIFICATION

The cleavage (transesterification) of polyribonucleotides is a process of considerable interest. The use of dinucleotide RNA fragments as su bstrates for the screening of RNA catalysis agents and mechanistic stu dies is widespread. This practice may not accurately predict the relat ive abilities of metal complexes to cleave polyribonucleotide substrat es. We report the use of chimeric DNA/RNA molecules, containing RNA nu cleotides embedded in DNA sequences, as substrates for studying the tr ansesterification of RNA. The substrates, termed embRNA, display the s implicity of dinucleotide substrates while possessing the multiple pho sphate and nucleobase metal-binding sites found in polyribonucleotides . In addition, the DNA residues provide an internal check for oxidativ e cleavage. The synthesis, purification, and activity of our first-gen eration embRNA, T(11)UT(7)A, is described. T(11)UT(7)A is a substrate for the ribonuclease RNase 1, and RNase 1 cleavage provides an excelle nt measure of the extent of 2'-deprotection in the synthetic embRNA. C leavage of T(11)UT(7)A by hydroxide and a variety of metal ions and co mplexes is also reported, and the use of embRNA in kinetic assays is d emonstrated. Competitive cleavage of RNA and DNA is built into the emb RNA assay. With Pb(II), Ce(III), and Cu(II) reagents, we observed effi cient RNA cleavage and no DNA cleavage. Kinetic comparison is made bet ween embRNA, T11UT7 and the analogous all-RNA substrate U-19.