The tyranny of adenosine recognition among RNA aptamers to coenzyme A

Abstract

Background: 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. 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.

Results: The 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.

Conclusion: Most 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.

Figure 1

Affinity matrices used in isolation of aptamers to adenosine cofactors. A) CoA attached to sepharose solid support through its pantotheine arm (disufide-linked CoA resin); B) CoA attached to sepharose through its primary amine at N6 (Amide-linked CoA resin); C) Other adenosine cofactors attached through the C8 of adenine. ATP, R = PPi; AMP, R = H, NAD⁺, R = nicotinamide mononucleotide.

Figure 2

Enrichment of aptamers to CoA. A) Black and gray columns represent the enrichment of 70SA and 80SA pools, respectively. White and striped columns bars represent the enrichment of 70PSA and 80PSA pools, respectively. Adenosine concentrations used during counter-selections for PSA pools are given beneath the graph. B) Sequences of aptamers that are used in experiments described in figure 3 and 4. Only the initially random portions are shown. Not shown are the 5' and 3' primer-binding sequences (see methods). Complete sequences of all aptamers from these selections are given.

Figure 3

Elution specificities of selected aptamers. A) The indicated aptamers were loaded onto the disulfide-linked CoA affinity resin. The percentage of input RNA eluted with 5 mM CoA are plotted on the graph. Prior to the elution, non-specifically-bound RNAs were removed with 10 column volumes of binding buffer (gray bars) or with 10 volumes of 1 mM adenosine (black bars). B) The aptamers indicated below each set of columns were bound to the disulfide-linked CoA resin, then washed with five volumes each of binding buffer, then 1 mM analog (gray bars), and finally of 5 mM CoA (black bars). Analogs were either pantotheine alone (P), AMP alone (A), AMP and pantotheine together (AP), or CoA (C). Percent elution is normalized to total RNA eluted in five "analog" fractions plus five "CoA" washes for ease of comparison. Under the conditions used in this assay, all of the aptamers gave approximately 50% total elution.

Figure 4

Refining the recognition specificity of four aptamers. Experimental design is as described in Figure 3. Analogs used in this assay are listed blow in the figure; Ade, adenosine; OAc, acetyl. The designation of each aptamer is indicated to the right of the corresponding panel.