Selection and Characterization of an RNA Decoy for Transcription Factor NF-κB

Abstract

Despite their chemical similarity, DNA and RNA sequences typically adopt very different structures within cells and are recognized by different proteins. However, a few interesting examples of proteins with dual specificity for DNA and RNA have previously been noted. These observations raise the possibility that RNA surrogates might be identified for many transcription factors that normally bind DNA. As an initial test of this novel concept, we used in vitro selection to isolate a small RNA aptamer that binds with nanomolar affinity to human transcription factor NF-kappa B, a key regulator of inflammation, HIV-1 gene expression, and apoptosis. Selected RNAs contain a 31-nucleotide core domain that was shown by mutation and deletion analyses to be necessary and sufficient for NF-kappa B binding. Neither DNA nor 2'-O-methyl RNA analogues of the aptamer bound NF-kappa B. The results of competition experiments demonstrate that binding of the RNA aptamer blocks the ability of NF-kappa B to bind duplex DNA. Expression of this aptamer structure within heterologous nuclear RNA transcripts may provide a new strategy to inhibit NF-kappa B function in vivo. Aptamers that inhibit transcription factors might be useful in a variety of applications.

Yeast genetic selections to optimize RNA decoys for transcription factor NF-κB

“…With the exception of one A-to-U mutation in RNA 2 (Fig. 2, red), all mutations occurred outside of the region previously shown to be important for (p50) 2 binding to the original in vitro-selected ␣-p50 aptamer (10). Interestingly, at three nucleotide positions, mutations occurred in two of the three selected sequences (Fig.…”

“…(p50) 2 and labeled RNA 1 or 6 were incubated in binding buffer (10 mM Hepes, pH 7.5͞100 mM NaCl͞1 mM DTT) for 30 min at 25°C, filtered over nitrocellulose membranes, and quantitated by scintillation counting. Estimates of K d were obtained by least-squares curve-fitting of binding isotherms (10).…”

“…Our laboratory previously conducted an in vitro selection seeking RNA ligands for NF-B, identifying a 31-nt RNA aptamer (␣-p50) that binds with high affinity (K d Ϸ 1 nM) to the target protein (10). Although selected for binding to the p50 homodimer form of NF-B, the ␣-p50 RNA aptamer also was shown to recognize the p50͞p65 heterodimer (10).…”

“…Although selected for binding to the p50 homodimer form of NF-B, the ␣-p50 RNA aptamer also was shown to recognize the p50͞p65 heterodimer (10). In addition, ␣-p50 functioned as an in vitro decoy for NF-B, competing with radiolabeled B DNA for transcription factor binding (10).…”

“…Although selected for binding to the p50 homodimer form of NF-B, the ␣-p50 RNA aptamer also was shown to recognize the p50͞p65 heterodimer (10). In addition, ␣-p50 functioned as an in vitro decoy for NF-B, competing with radiolabeled B DNA for transcription factor binding (10). Furthermore, the ␣-p50 RNA aptamer was shown to recognize its (p50) 2 target within a eukaryotic nucleus (11) in a yeast three-hybrid assay (12).…”