Target specificity and structural characterization of single-stranded DNA aptamer RT1t49, a broad inhibitor of HIV-1 reverse transcriptases

Single-stranded (ss) DNA and RNA aptamers have been selected to the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) protein. These aptamers bind RT in the mid-picomolar to high-nanomolar range and are potent inhibitors of the polymerase and RNase H functions of RT. While a substantial body of work has been directed towards understanding the essential structural features of RNA aptamers and their interactions with RT, there has been much less work devoted to the overall structural requirements and interactions of ssDNA aptamers with RT. This project focused on developing a thorough understanding of the structural requirements for ssDNA aptamer RT1t49-RT binding and established RT1t49 as a broad inhibitor of HIV-1, HIV-2, and SIVCPZ RTs. Three structural domains were found to be essential for RT inhibition by RT1t49: a 5' stem (stem I), a connector and a 3' stem (stem II) capable of forming multiple secondary structures. Hydroxyl radical cleavage and structural mapping support the model that this aptamer is binding as a primer-template mimic with nucleotide A32 making close contacts to the polymerase active site and the variable stem II potentially making contacts with the back-side of the fingers domain. Pre-steady-state and order-of-addition kinetic analyses established that this ssDNA aptamer competes with primer-template for access to RT. Enzymatic extension assays and RNase H cleavage assays demonstrated this aptamer inhibits RT activity from HIV-1 M subtypes B, D, C, A, and G as well as RT from HIV-1 group O, SIVCPZ and HIV-2. Transfection of RT1t49 with HIV-encoding plasmids reduced viral infection in the subsequent round by ∼80% and Oligofectamine-mediated transfection of RT1t49 reduced HIV-1 infectivity by ∼60%. Overall, the work presented in this thesis highlights the utility of this class of molecules as novel therapeutic potentials for treatment of HIV-1.