La. Jenkins et al., THE EMBEDDED RIBONUCLEOTIDE ASSAY - A CHIMERIC SUBSTRATE FOR STUDYINGCLEAVAGE OF RNA BY TRANSESTERIFICATION, Journal of the American Chemical Society, 118(29), 1996, pp. 6822-6825
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.