Abstract
Multiple targets exist in the development of HIV-1 anti-viral drugs, one of which includes the interaction between the transcriptional activator protein (Tat) and the Trans Activation Response Region (TAR) element of RNA. During transcription, TAR RNA, a 59-base stem-bulge-loop structure, located at the 5’end of all HIV-1 mRNAs, recruits Tat, which modulates viral gene expression in infected cells. Previous experiments have shown that the Arginine Rich Motif (ARM) of Tat is integral for the association of Tat to TAR. Altogether, literature suggests that the inhibition of Tat/TAR RNA interaction is an attractive route to controlling HIV-1 expression and replication. We sought to design synthetic polymers that would disrupt the necessary interaction between Tat and TAR RNA, hindering HIV replication. To target the TAR-RNA, we sought to replicate the basic ARM of Tat by functionalizing amine-amenable polymer scaffolds derived from the Ring-Opening Metathesis Polymerization (ROMP), with the guanidinium derivatives of arginine and agmatine. To this end, we have successfully synthesized the guanidinylated polymers. We hypothesize that by amending the polymer scaffolds with guanidinums, an essential requisite for binding of small molecules to TAR RNA, we may inhibit the mobility shift of the RNA. To test this hypothesis, we assayed the RNA-binding activities of the guanidinylated polymers using an Electrophoretic Mobility Shift Assay (EMSA)—based approach. Our studies indicate that all the synthetic guandiniums are RNA-binding molecules that recognize and retard the mobility of wild-type TAR RNA in a concentration range of 1—400 μM. Additionally, we found that the introduction of magnesium (Mg2+) in the binding buffer strongly stimulates RNA folding as well as increases the RNA-binding specificity of the polymeric compounds. To optimize the binding between the polymers and the RNA we will explore different binding buffers that may increase the binding affinity, allowing us to characterize the Ka and Kd values. In addition, it is expected that TAR-RNA binding molecules may inhibit the association of Tat/TAR; therefore, ongoing work seeks to elucidate the selectivity and specificity of the guanidinum-conjugated polymers, in addition determining the effects of the polymers on protein-TAR RNA interactions by EMSA.