A novel acidophilic RNA motif that recognizes coenzyme A.
Specific recognition of nucleotide cofactors by RNA may be important in engineering new RNA enzymes (ribozymes). Although in vitro selections (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 aptamers that bind CoA. Functional boundary determination and extensive comparative sequence analysis (including reselection of a mutagenized, circularly permuted RNA) led to the identification of a 52 nucleotide minimal aptamer ("min52"). The RNA structural motif contains a large internal loop with 26 unpaired nucleotides flanked by helices of any base-paired sequence. Twenty loop nucleotides are specifically required for binding activity, 12 of which are derived from the original primer binding sequences. Specificity studies with CoA analogues demonstrated that the aptamer recognizes many adenosine analogues, including ATP, and that recognition is predominantly through the Höogsteen 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 Mg2+ 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 evolution of life. In the presence of 10 mM Mg2+, binding is unaffected by the addition of 1 mM Ca2+, but it is mildly inhibited by 1 mM Zn2+ or Co2+ or by 0.1 mM Cu2+ or Ni2+. The dissociation constant (Kd) for the association of min52 RNA with ATP in solution was measured to be 2.4 +/- 0.4 microM under the conditions of the selection and 0.5 +/- 0.1 microM under optimized conditions. Finally, we show that the selected CoA aptamer populations contain other RNAs at low frequencies that preferentially recognize intact CoA and are not eluted from the resin by AMP alone.
Burke
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DH
Department of Molecular, Cellular, & Developmental Biology, Campus Box 347, University of Colorado, Boulder, Colorado 80309-0347, USA. dhburke@beagle.colorado.edu
Hoffman
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DC
D000241:Adenosine / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003065:Coenzyme A / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011119:Polynucleotides / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012996:Solutions; D014158:Transcription, Genetic
D011119:Polynucleotides; D012996:Solutions; D012313:RNA; D000255:Adenosine Triphosphate; D000241:Adenosine; D003065:Coenzyme A