An RNA Aptamer with High Affinity and High Specificity for the 5S RNA Binding Zinc Finger Proteins TFIIIA and p43

Abstract

The Xenopus zinc finger proteins TFIIIA and p43 bind to 5S RNA in immature oocytes to form 7S and 42S ribonucleoprotein storage particles. To probe the similarities and differences in the RNA binding domains of these two proteins, a library of random RNA molecules was enriched using TFIIIA as the bait protein. One of the abundant aptamers isolated, RNA22, bound to both TFIIIA and p43 derived zinc finger peptides with high affinity and specificity even though the predicted secondary structure of the RNA was unrelated to that of 5S RNA. The interactions of TFIIIA and p43 peptides with RNA22 were compared to their interactions with 5S RNA by characterizing the effects of assay conditions, mutations in RNA22, and mutations in the zinc finger proteins. The similarities and differences in the mechanisms by which these two zinc finger proteins interact with 5S RNA compared to RNA22 suggest they share a common platform for RNA binding with enough flexibility to form specific interactions with both RNAs.

An RNA Aptamer with High Affinity and Broad Specificity for Zinc Finger Proteins

“…Basic Requirements of Zinc Finger Binding to RNA1. RNA1 (Figure 1A) was the most abundant aptamer selected from a random library of RNA molecules using the bait protein TFIIIA (31) and contains the GGGUGGG consensus motif found in two-thirds of the aptamers in the enriched library. RNA1 shows virtually no specificity for a particular zinc finger protein: all of the zinc finger proteins tested exhibited high affinity for RNA1.…”

“…To understand how RNA1 might act as a broad-spectrum ligand for zinc finger proteins, we compared the effects of different binding conditions on the interaction of the three most abundant RNA aptamers (RNA1, RNA21, and RNA22) with the original bait protein TFIIIA. These aptamers have high affinities for TFIIIA but differ in the degree of specificity they have for this protein compared to any other zinc finger protein (31).…”

“…Our initial intent in selecting RNA aptamers with high affinity for TFIIIA was to use them to test whether the related 5S rRNA binding protein p43 shared a common structural framework for RNA binding. The three most abundant sequences in the enriched library of RNA aptamers all had similar high affinities for TFIIIA but exhibited distinct characteristics in terms of their degree of specificity for binding TFIIIA compared to other zinc finger proteins (31). RNA1 exhibited a complete lack of specificity for TFIIIA, binding with subnanomolar to nanomolar affinities to all of the C 2 H 2 zinc finger proteins tested.…”

“…Construction of Zinc Finger Protein Expression Vectors. Construction of plasmid vectors used to express Xenopus TFIIIA and the zinc finger peptides YY1 and ZFY6 has been described previously (31).…”

“…Given these differences in the conformations of DNA and RNA, it is remarkable that at least some zinc finger proteins are able to form specific interactions with both classes of nucleic acids. After enriching a library of random RNA sequences for aptamers that form high-affinity complexes with TFIIIA, we determined that the two most abundant aptamers demonstrated high affinity for zinc finger proteins in general (31). One of these aptamers (RNA1) bound to all zinc finger proteins that we tested with affinities in the subnanomolar range.…”