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

摘要

The cleavage (transesterification) of polyribonucleotides is a process of considerable interest. The use of dinucleotide RNA fragments as substrates for the screening of RNA catalysis agents and mechanistic studies is widespread. This practice may not accurately predict the relative abilities of metal complexes to cleave polyribonucleotide substrates. We report the use of chimeric DNA/RNA molecules, containing RNA nucleotides embedded in DNA sequences, as substrates for studying the transesterification of RNA. The substrates, termed embRNA, display the simplicity of dinucleotide substrates while possessing the multiple phosphate and nucleobase metal-binding sites found in polyribonucleotides. In addition, the DNA residues provide an internal check for oxidative cleavage. The synthesis, purification, and activity of our first-generation 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 excellent measure of the extent of 2'-deprotection in the synthetic embRNA. Cleavage of T(11)UT(7)A by hydroxide and a variety of metal ions and complexes is also reported, and the use of embRNA in kinetic assays is demonstrated. Competitive cleavage of RNA and DNA is built into the embRNA assay. With Pb(II), Ce(III), and Cu(II) reagents, we observed efficient RNA cleavage and no DNA cleavage. Kinetic comparison is made between embRNA, T11UT7 and the analogous all-RNA substrate U-19.