DNA binding transcription factors are integral to transcription activation and repression and in many cases are comprised of multiple domains and subunits. To elucidate the contributions of these subunits to the interactions that these factors make, surface specific inhibitors must be developed and characterized which have the potential to block a specific domain of the factor thereby impairing its activity. The Heat Shock Response is a highly conserved protective mechanism that is regulated at the transcriptional level by transcription factors called Heat Shock Factors (HSFs). When activated by high temperatures or proteotoxic stress, HSFs strongly induce the expression of Heat Shock genes (HS genes), that encode Heat Shock Proteins (HSPs). Of the family of HSFs in mammals, HSF1 is the functional homolog of the single HSF in yeast and fruit fly. HSF1 consists of at least five major domains; the DNA Binding Domain (DBD), Trimerization Domain (TD), the Regulatory Domain (RD), the Leucine Zipper Regulatory Domain (HR-C) and the Trans-Activation Domain (TAD) which function to coordinate HSF1’s ability to bind to its target DNA elements and trans-activate HS genes under heat shock conditions. To elucidate the mechanistic roles of each domain in living cells, I have used the novel approach of blocking domains with RNA aptamers. RNA aptamers are short, single stranded RNAs that bind specifically and with high affinity to their selected target, whether it is a protein domain, transcription factor or small molecule. We have thousands of selected candidate aptamers for HSF1, however, we needed to identify those that have domain specific binding in order to test a domain specific effect. Initially, to identify the binding affinities of the selected RNA Aptamers, I used Electrophoretic Mobility Shift Assays (EMSA). As part of this study, I have screened aptamers that are able to bind hHSF1 strongly and identified the binding regions of some high-affinity candidates. Many of the aptamers directly interact with a minimal hHSF1 containing the regulatory, trimerization and DNA-binding domains. Of the high-affinity binders only the top-aptamer partially competes with hHSF1 for binding to a heat shock element. To identify a genome-wide effect of the characterized top aptamer which has some competitive ability, I performed a PRO-seq experiment. We tried this experiment multiple times but were unable to measure a genome-wide effect. To improve the ability of these RNA aptamers as molecular inhibitors, I used RNA scaffolds to generate multimeric aptamers using the minimal core aptamers. Dimerization and trimerization enhances the competitive behavior of the monomeric aptamers up to 70-fold. Overall, we have generated and optimized high-affinity tools to interfere with the DNA-binding activity of hHSF1 that provide further opportunities for precisely disrupting the functions of hHSF1 in a biological context.