Abstract
BackgroundUnderstanding the diversity of interactions between RNA aptamers and nucleotide cofactors promises both to facilitate the design of new RNA enzymes that utilize these cofactors and to constrain models of RNA World evolution. In previous work, we isolated six pools of high affinity RNA aptamers to coenzyme A (CoA), the principle cofactor in biological acyltransfer reactions. Interpretation of the evolutionary significance of those results was made difficult by the fact that the affinity resin attachment strongly influenced the outcome of those selections. Here we describe the selection of four new pools isolated on a disulfide-linked CoA affinity matrix to minimize context-dependent recognition imposed by the attachment to the solid support.ResultsThe four new aptamer libraries show no sequence or structural relation to a previously dominant CoA-binding species, even though they were isolated from the same initial random libraries. Recognition appears to be limited to the adenosine portion of the CoA – in particular the Höogsteen edge – for most isolates surveyed, even when a counter selection was employed to remove such RNAs. Two of the recovered isolates are eluted with intact CoA more efficiently than with AMP alone suggesting a possible pantotheine interaction. However, a detailed examination of recognition specificity revealed that the 3' phosphate of CoA, and not the pantotheine arm, determined recognition by these two isolates.ConclusionMost aptamers that have been targeted towards cofactors containing adenosine recognize only the adenosine portion of the cofactor. They do not distinguish substituents on the 5' carbon, even when those substituents have offered hydrogen bonding opportunities and the selection conditions discouraged adenosine recognition. Beyond hydrogen bonding, additional factors that guide the selection towards adenosine recognition include aromatic stacking interactions and relatively few rotational degrees of freedom. In the present work, a sterically accessible, disulfide-linked CoA affinity resin afforded the selection of a more diverse aptamer collection then previous work with a N6 linked CoA resin.
Highlights
Understanding the diversity of interactions between RNA aptamers and nucleotide cofactors promises both to facilitate the design of new RNA enzymes that utilize these cofactors and to constrain models of RNA World evolution
We find that while the requirements for recognition for most aptamers are fully satisfied with adenine alone, a few aptamers require the 3' phosphate found in coenzyme A (CoA), but none is shown unambiguously to make contacts with the pantotheine
Selection of SA and PSA pools To evaluate the effect of affinity resin linkage on the selection of CoA aptamers and to obtain new libraries of CoAbinding RNA aptamers, CoA was immobilized on an activated thiopropyl sepharose matrix through a disulfide bond to its unique sulfur
Summary
Understanding the diversity of interactions between RNA aptamers and nucleotide cofactors promises both to facilitate the design of new RNA enzymes that utilize these cofactors and to constrain models of RNA World evolution. We isolated six pools of high affinity RNA aptamers to coenzyme A (CoA), the principle cofactor in biological acyltransfer reactions. Interpretation of the evolutionary significance of those results was made difficult by the fact that the affinity resin attachment strongly influenced the outcome of those selections. We describe the selection of four new pools isolated on a disulfide-linked CoA affinity matrix to minimize context-dependent recognition imposed by the attachment to the solid support
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