Nucleic Acids Research Advance Access published online on March 20, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp177
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A role for hydrophobicity in a Diels–Alder reaction catalyzed by pyridyl-modified RNA
1Department of Molecular and Structural Biochemistry and 2Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA
*To whom correspondence should be addressed. Tel: +1 919 515 8915; Fax: +1 919 515 8920; Email: stefan_franzen{at}ncsu.edu
Received December 20, 2008. Revised March 1, 2009. Accepted March 2, 2009.
New classes of RNA enzymes or ribozymes have been obtained by in vitro evolution and selection of RNA molecules. Incorporation of modified nucleotides into the RNA sequence has been proposed to enhance function. DA22 is a modified RNA containing 5-(4-pyridylmethyl) carboxamide uridines, which has been selected for its ability to promote a Diels–Alder cycloaddition reaction. Here, we show that DA_TR96, the most active member of the DA22 RNA sequence family, which was selected with pyridyl-modified nucleotides, accelerates a cycloaddition reaction between anthracene and maleimide derivatives with high turnover. These widely used reactants were not used in the original selection for DA22 and yet here they provide the first demonstration of DA_TR96 as a true multiple-turnover catalyst. In addition, the absence of a structural or essential kinetic role for Cu2+, as initially postulated, and nonsequence-specific hydrophobic interactions with the anthracene substrate have led to a reevaluation of the pyridine modification's role. These findings broaden the catalytic repertoire of the DA22 family of pyridyl-modified RNAs and suggest a key role for the hydrophobic effect in the catalytic mechanism.
Present address: Keith T. Gagnon, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA