The Embedded Ribonucleotide Assay: A Chimeric Substrate for Studying Cleavage of RNA by Transesterification Lisa A. Jenkins, James K. Bashkin,* ,† and Mark E. Autry Contribution from the Department of Chemistry, Washington UniVersity, Campus Box 1134, 1 Brookings DriVe, St. Louis Missouri 63130 ReceiVed January 16, 1996 X Abstract: 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 T 11 UT 7 and the analogous, all-RNA substrate U 19 . Many groups are actively developing RNA cleavage agents with possible medical applications, including the gene-specific, catalytic destruction of viral mRNA. 1-19 Generally, catalysts are chosen or rejected based on their ability to cleave dinucle- otide substrates, 20-23 although we and others have reported studies based on metal-promoted cleavage of RNA oligomers and polymers. 9,24-26 The dinucleotide assays may provide misleading information about polynucleotide cleavage, due to the length-dependence of the RNA transesterification reaction (Vide infra). Polymeric substrates themselves present a multi- plicity of reaction sites that may not be kinetically equivalent. Furthermore, the products of each polymer cleavage reaction are substrates for further reaction, which can complicate kinetic studies beyond the initial rate regime. To overcome these limitations and to allow unprecedented control over the se- quence and electrostatic context of RNA cleavage, we report a new assay for RNA transesterification that allows (1) a simplified study of high MW polynucleotide substrates, (2) an internal check for oxidative cleavage processes, (3) a systematic exploration of the sequence context effects on RNA cleavage, and (4) the study of competition between RNA and DNA cleavage. We have named this assay the Embedded Ribonucle- otide Assay. It employs chimeric oligonucleotides that contain one or more RNA nucleotides inserted at controlled positions into DNA sequences. We use enzymatic cleavage of the embedded RNA (embRNA) to demonstrate biological activity and complete deprotection of the 2′-OH groups in our chemi- cally-synthesized substrate. Our first embRNA substrate con- sists of RNA embedded into unmodified DNA, but the method is general and allows incorporation of DNA modifications at specific sites, including methylphosphonate linkages, deaza- or methylated nucleobases, or phosphorothioates. We show the first examples of competitive cleavage of RNA and DNA by † Phone: (314) 935-4801, FAX (314) 935-4481. Email: Bashkin@ wuchem.wustl.edu. X Abstract published in AdVance ACS Abstracts, July 1, 1996. (1) Bashkin, J. K.; Frolova, E. I.; Sampath, U. J. Am. Chem. Soc. 1994, 116, 5981-5982. (2) Bashkin, J. K.; Jenkins, L. A. Comments Inorg. Chem. 1994, 16, 77-93. (3) Bashkin, J. K.; Sondhi, S. M.; Modak, A. S.; Sampath, U.; d’Avignon, D. A. New J. Chem. 1994, 18, 305-316. (4) Bashkin, J. K.; Sampath, U. S.; Frolova, E. I. Appl. Biochem. Biotechnol. 1995, 54, 43-56. (5) Bashkin, J. K.; Jenkins, L. A. J. Chem. Soc., Dalton Trans. 1993, 3631. (6) Bashkin, J. K. Bioinorganic Chemistry of Copper; Karlin, K. D., Tyeklar, Z., Eds.; Chapman and Hall: New York, 1993; pp 132-139. (7) Bashkin, J. K.; McBeath, R. J.; Modak, A. S.; Sample, K. R.; Wise, W. B. J. Org. Chem. 1991, 56, 3168. (8) Modak, A. S.; Gard, J. K.; Merriman, M. C.; Winkeler, K. A.; Bashkin, J. K.; Stern, M. K. J. Am. Chem. Soc. 1991, 113, 283. (9) Stern, M. K.; Bashkin, J. K.; Sall, E. D. J. Am. Chem. Soc. 1990, 112, 5357. (10) Bashkin, J. K.; Gard, J. K.; Modak, A. S. J. Org. Chem 1990, 55, 5125. (11) Bashkin, J. K.; Xie, J.; Daniher, A. T.; Jenkins, L. A.; Yeh, G. C. Ribozyme Mimics for Catalytic Antisense Strategies; Meunier, B., Ed.; Kluwer: Netherlands, 1996; pp 355-366. (12) Bashkin, J. K.; Xie, J.; Daniher, A. T.; Sampath, U.; Kao, J. L.-F. J. Org. Chem. 1996, 61, 2314-2321. (13) Bashkin, J. K.; Sampath, U. S.; Frolova, E. I. Appl. Biochem. Biotechnol. 1995, 54, 43-56. (14) Komiyama, M.; Inokawa, T.; Yoshinari, K. J. Chem. Soc., Chem. Commun. 1995, 77-78. (15) Morrow, J. R.; Shelton, V. M. New J. 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