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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.

The hairpin ribozyme catalyzes a reversible phosphodiester cleavage reaction. We examined the roles of conserved nucleobases in catalysis using an abasic ribozyme rescue strategy. Loss of the active site G8 nucleobase reduced the cleavage rate constant by 350-fold while loss of A9 and A10 nucleobases reduced activity less than 10-fold. Certain heterocyclic amines restored partial activity when provided in solution to the variant lacking G8. Heterocyclic amines that were capable of rescue shared the exocyclic amine and cyclic amide in common with the Watson-Crick hydrogen bonding face of guanine. In contrast to the shallow pH dependence of unmodified ribozyme activity, rescue activity increased sharply with decreasing pH. These results support a novel model for RNA catalysis in which a cationic nucleobase contributes electrostatic stabilization to negative charge developing in the transition state.

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