Aptamers to Chloramphenicol

Chloramphenicol is derived from the biosynthesis pathway of aromatic amino acids like phenylalanine and targets the peptidyltransferase center in the 23S ribosomal RNA. It inhibits peptide bond formation by binding to the peptidyl-transferase loop of the 23S rRNA in the large ribosomal subunit. It is believed that chloramphenicol acts as a conformational analog of the 3'-O(aminoacyl)ade-nosine part of the incoming aminoacyl-tRNA (Bhuta et al., 1980; Drainas et al., 1993; Zemlicka et al., 1993) or of the transition state for peptide bond formation or a newly formed peptide bond (Bhuta et al., 1980). This implies that the interaction of chloramphenicol with the ribosome is somehow similar to the interaction of the ribosome with a component of the translational machinery. As the pep-tidyltransferase loop participates in peptide bond formation and chloramphenicol recognizes a part of the rRNA involved in this process, aptamers against chloram-phenicol were selected in order to learn about how rRNA contributes to the formation of peptide bonds (Burke, 1997).


In order to select aptamers against chloramphenicol, two individual RNA pools were used. They consisted of 70 and 80 randomized nucleotides respectively, containing 1014-1015 sequences. For the selection procedure an agarose resin was de-rivatized with chloramphenicol and antibiotic-binding RNAs were affinity eluted. Elution required long incubation times in order to bias the selection towards high-affinity aptamers with long feoff rates. While the recovery of specific RNAs was low in the first seven cycles, it increased up to 50% in the 12th cycle, with Kd-values of 25 mmol/L (70 nt pool) and 65 mmol/L (80 nt pool). RNAs from both pools contained the sequence motif shown in Table 5.1, consisting of three helices interrupted by two asymmetric bulges with 4-6 adenosines across from a single adenosine. This motif was identified by determining the 3' and 5' boundaries of the sequence required for chloramphenicol binding, RNase S1 analysis and investigation of the activity of truncated RNAs. For the latter assay a 50-nt minimal RNA (Cm1) containing only the identified motif, as well as a 33-nt RNA (Cm2) containing only one bulge, that is one half of the otherwise symmetrical motif (Table 5.1), were synthesized. While Cm2 showed little activity, Cm1 displayed activities similar to those of the full-length RNAs. Thus, Cm1 was resynthesized with a 15% mutagenesis rate per position and subjected to reselection in order to further refine the sequence requirements for chloramphenicol binding. Moreover, the stoichiometry of the bound complex was analyzed by NMR spectroscopy and found to be 1:1. Therefore, two models for binding of chloramphenicol to its aptamer are possible. Either the two symmetric halves of the selected structure come together through tertiary interactions and form a single binding site, or the aptamer contains two identical binding sites which can be occupied only one at a time. As Cm2, corresponding to such a single binding site, shows only little affinity to chloramphenicol, the first possibility seems to be more likely.

By comparing the sequence of the peptidyltransferase loop of the 23S rRNA with the sequence of the selected aptamer, certain parallels become obvious. By juxtaposing three arcs of the peptidyltransferase loop, it is possible to model a binding site similar to the selected one, containing nucleotides actually involved in chloramphenicol binding. However, this model is based on the independence of the two halves of Cm1, and therefore made assailable by the low activity of Cm2.

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