A NOVEL ACIDOPHILIC RNA MOTIF THAT RECOGNIZES COENZYME-A

Specific recognition of nucleotide cofactors by RNA may be important i n engineering new RNA enzymes (ribozymes). Although in vitro selection s (SELEX) have identified nucleic acid motifs (''aptamers'') that bind a variety of adenosine cofactors, none of these recognizes coenzyme A (CoA), the primary biological cofactor used in acyltransfer reactions . We used SELEX experiments with two random RNA pools to identify apta mers that bind CoA. Functional boundary determination and extensive co mparative sequence analysis (including reselection of a mutagenized, c ircularly permuted RNA) led to the identification of a 52 nucleotide m inimal aptamer (''min52''). The RNA structural motif contains a large internal loop with 26 unpaired nucleotides flanked by helices of any b ase-paired sequence. Twenty loop nucleotides are specifically required for binding activity, 12 of which are derived from the original prime r binding sequences. Specificity studies with CoA analogues demonstrat ed that the aptamer recognizes many adenosine analogues, including ATP , and that recognition is predominantly through the Hoogsteen face of adenine. Binding activity is greatest at acidic pH (optimum near 5.0), in low or no monovalent salt, and at high concentrations of either Mg 2+ or Mn2+. Strong binding activity (86% of maximum) is observed at pH 4.0, suggesting that at least some extreme conditions (acidic pH) may be compatible with RNA World theories of the origin and early evoluti on of life. In the presence of 10 mM Mg2+, binding is unaffected by th e addition of 1 mM Ca2+, but it is mildly inhibited by 1 mM Zn2+ or Co 2+ or by 0.1 mM Cu2+ or Ni2+. The dissociation constant (K-d) for the association of min52 RNA with ATP in solution was measured to be 2.4 /- 0.4 mu M under the conditions of the selection and 0.5 +/- 0.1 mu M under optimized conditions. Finally, we show that the selected CoA ap tamer populations contain other RNAs at low frequencies that preferent ially recognize intact CoA and are not eluted from the resin by AMP al one.