ABSTRACT
Macromolecular interactions are central to the regulation and execution of many key biological processes, and therefore, they are attractive targets for drug discovery. Previously, we identified an RNA aptamer for the heat shock factor (HSF1), which is capable of interfering with the binding of HSF1 to its cognate DNA elements. Here we report the significant enhancement of avidity through dimerization of this aptamer. In particular, we describe the effect of 2 factors in designing a multivalent aptamer: the distance between active subunits and the flexibility of the linkage.
Subject(s)
Aptamers, Nucleotide/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA/metabolism , Drosophila Proteins/antagonists & inhibitors , Drosophila melanogaster/metabolism , Heat-Shock Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae/metabolism , Transcription Factors/antagonists & inhibitors , Animals , Aptamers, Nucleotide/chemical synthesis , Aptamers, Nucleotide/genetics , Base Sequence , DNA/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimerization , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Heat Shock Transcription Factors , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , Quantitative Structure-Activity Relationship , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
We developed a powerful expression system to produce aptamers and other types of functional RNA in yeast to examine their effects. Utilizing the intron homing process, the aptamer-coding sequences were integrated into hundreds of rRNA genes, and the aptamers were transcribed at high levels by RNA polymerase I without any additional promoter being introduced into the cell. We used this system to express an aptamer against the heat shock factor 1 (HSF1), a conserved transcription factor responsible for mobilizing specific genomic expression programs in response to stressful conditions such as elevated temperature. We observed a temperature sensitive growth retardation phenotype and specific decrease of heat shock gene expression. As HSF1 enables and promotes malignant growth and metastasis in mammals, and this aptamer binds yeast HSF1 and its mammalian ortholog with equal affinity, the results presented here attest to the potential of this aptamer as a specific and effective inhibitor of HSF1 activity.
Subject(s)
Aptamers, Nucleotide/metabolism , DNA-Binding Proteins/antagonists & inhibitors , Gene Expression Regulation , Introns , Transcription Factors/antagonists & inhibitors , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/genetics , Gene Knockdown Techniques , Heat Shock Transcription Factors , Phenotype , RNA/chemistry , Saccharomyces cerevisiae/geneticsABSTRACT
Heat shock factor (HSF) is a conserved and highly potent transcription activator. It is involved in a wide variety of important biological processes including the stress response and specific steps in normal development. Reagents that interfere with HSF function would be useful for both basic studies and practical applications. We selected an RNA aptamer that binds to HSF with high specificity. Deletion analysis defined the minimal binding motif of this aptamer to be two stems and one stem-loop joined by a three-way junction. This RNA aptamer interferes with normal interaction of HSF with its DNA element, which is a key regulatory step for HSF function. The DNA-binding domain plus a flanking linker region on the HSF (DL) is essential for the RNA binding. Additionally, this aptamer inhibits HSF-induced transcription in vitro in the complex milieu of a whole cell extract. In contrast to the previously characterized NF-kappaB aptamer, the HSF aptamer does not simply mimic DNA binding, but rather binds to HSF in a manner distinct from DNA binding to HSF.
