Gagnon Keith T, Ju Show-Yi, Goshe Michael B, Maxwell E Stuart, Franzen Stefan
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.
Nucleic Acids Res. 2009 May;37(9):3074-82. doi: 10.1093/nar/gkp177. Epub 2009 Mar 20.
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 Cu(2+), 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.
通过对RNA分子进行体外进化和筛选，已获得了新型的RNA酶或核酶。有人提出将修饰的核苷酸掺入RNA序列中以增强其功能。DA22是一种含有5-(4-吡啶甲基)羧酰胺尿苷的修饰RNA，它因其促进狄尔斯-阿尔德环加成反应的能力而被筛选出来。在此，我们表明，DA_TR96是DA22 RNA序列家族中活性最高的成员，它是用吡啶修饰的核苷酸筛选出来的，能以高周转率加速蒽与马来酰亚胺衍生物之间的环加成反应。这些广泛使用的反应物在最初筛选DA22时并未使用，但在此它们首次证明了DA_TR96是一种真正的多周转催化剂。此外，最初假设的Cu(2+)不存在结构或关键动力学作用，以及与蒽底物的非序列特异性疏水相互作用，导致人们对吡啶修饰的作用进行了重新评估。这些发现拓宽了吡啶修饰的DA22家族RNA的催化范围，并表明疏水效应在催化机制中起关键作用。
