Rational design of a structure-switching DNA aptamer for potassium ions.

Structure-switching molecules provide a unique means for analyte detection, generating a response to analyte concentration through a binding-specific conformational change between non-binding and binding-competent states. While most ligand-binding molecules are not structure switching by default, many can be engineered to be so through the introduction of an alternative non-binding (and thus non-signalling) conformation. This population-shift mechanism is particularly effective with oligonucleotides and has led to the creation of structure-switching aptamers for many target ligands. Here, we report the rational design of structure-switching DNA aptamers, based on the thrombin binding aptamer (TBA), that bind potassium with affinities that bridge the gap between previously reported weak-binding and strong-binding aptamers. We also demonstrate a correlation between the free energy of the experimentally determined binding affinity for potassium and the computationally estimated free energy of the alternative (non-binding) structure.

RNA dimerization promotes PKR dimerization and activation.

The double-stranded RNA (dsRNA)-activated protein kinase [protein kinase R (PKR)] plays a major role in the innate immune response in humans. PKR binds dsRNA non-sequence specifically and requires a minimum of 15-bp dsRNA for one protein to bind and 30-bp dsRNA to induce protein dimerization and activation by autophosphorylation. PKR phosphorylates eukaryotic initiation factor 2alpha, a translation initiation factor, resulting in the inhibition of protein synthesis. We investigated the mechanism of PKR activation by an RNA hairpin with a number of base pairs intermediate between these 15- to 30-bp limits: human immunodeficiency virus type 1 transactivation-responsive region (TAR) RNA, a 23-bp hairpin with three bulges that is known to dimerize. TAR monomers and dimers were isolated from native gels and assayed for RNA and protein dimerization to test whether RNA dimerization affects PKR dimerization and activation. To modulate the extent of dimerization, we included TAR mutants with different secondary features. Native gel mixing experiments and analytical ultracentrifugation indicate that TAR monomers bind one PKR monomer and that TAR dimers bind two or three PKRs, demonstrating that RNA dimerization drives the binding of multiple PKR molecules. Consistent with functional dimerization of PKR, TAR dimers activated PKR while TAR monomers did not, and RNA dimers with fewer asymmetrical secondary-structure defects, as determined by enzymatic structure mapping, were more potent activators. Thus, the secondary-structure defects in the TAR RNA stem function as antideterminants to PKR binding and activation. Our studies support that dimerization of a 15- to 30-bp hairpin RNA, which effectively doubles its length, is a key step in driving activation of PKR and provide a model for how RNA folding can be related to human disease.

The double-stranded-RNA-binding motif: interference and much more.

RNA duplexes have been catapulted into the spotlight by the discovery of RNA interference and related phenomena. But double-stranded and highly structured RNAs have long been recognized as key players in cell processes ranging from RNA maturation and localization to the antiviral response in higher organisms. Penetrating insights into the metabolism and functions of such RNAs have come from the identification and study of proteins that contain the double-stranded-RNA-binding motif.

A mechanistic framework for co-transcriptional folding of the HDV genomic ribozyme in the presence of downstream sequence.

Hepatitis delta virus (HDV) is a circular pathogenic RNA that uses self-cleavage by closely related 84 nt genomic and antigenomic ribozymes to facilitate the replication of its genome. Downstream of each ribozyme is a stretch of nucleotides termed the attenuator that functions to base-pair with and unfold the ribozyme into a rod-like fold. The competing rates of RNA synthesis, ribozyme folding and cleavage, and rod folding are therefore likely to affect the efficiency of co-transcriptional self-cleavage. In these studies, co-transcriptional folding of the genomic ribozyme was assayed in vitro by monitoring co-transcriptional self-cleavage of transcripts having variable lengths of sequence downstream of the ribozyme. Co-transcriptional cleavage data were simulated successfully only with kinetic models in which cleavage-inactive channels were populated during transcription. Partitioning to and escape from these channels was influenced, in part, by whether the available attenuator sequence could form structures with the ribozyme, and by the stability of such structures. Surprisingly, only 23 nt of attenuator were needed for strong inactivation of cleavage. Self-cleavage of certain 3'-virus-containing sequences could be restored, partially, by renaturation; however, self-cleavage of transcripts with a full-length attenuator could not be restored efficiently by renaturation in vitro. This suggests that in the presence of the attenuator, the cleavage-active ribozyme fold is not the thermodynamically most stable species. In accordance with this model, the efficiency of self-cleavage of the ribozyme followed by a full-length attenuator was increased by decreasing the rate of transcription. These results suggest that, in the absence of additional factors, efficient co-transcriptional cleavage of the full-length genomic HDV RNA may require cleavage to occur prior to synthesis of the attenuator.