A novel RNA motif that binds efficiently and specifically to the Tat protein of HIV and inhibits the trans-activation by Tat of transcription in vitro and in vivo

Abstract

Background: To find a novel RNA that would bind efficiently and specifically to Tat protein but not to other cellular factors, we used an in vitro selection method and isolated a novel aptamer RNA(Tat), a 37-mer RNA oligomer, that binds efficiently to the Tat protein of HIV-1. In the present study, we analysed various properties of aptamer RNA(Tat), including binding kinetics, identification of functional groups for Tat binding, and inhibition of Tat function. Results: The binding affinity of the isolated aptamer RNA(Tat) to Tat-1 was 133 times higher than that of authentic TAR-1 RNA. RNA(Tat) is composed of inverted repeats of two TAR-like motifs, and even though RNA(Tat) had two Tat-binding core elements, the interaction with Tat took place at a molar ratio of 1:1. Several functional groups of aptamer RNA(Tat) responsible for Tat binding were identified. The selected aptamer RNA(Tat) competed effectively for binding to Tat even in the presence of a large excess of TAR-1 or TAR-2 RNA in vitro, and specifically prevented Tat- dependent trans-activation both in vitro and in vivo. Conclusions: Our results indicate that a novel aptamer, RNA(Tat), retained strong affinity for Tat even in the presence of a large excess of HIV TAR. RNA(Tat) binds efficiently to Tat proteins or peptides derived from either HIV-1 or HIV-2. Unlike TAR RNA, RNA(Tat) affinity does not depend upon cellular proteins such as cyclin T1, thus RNA(Tat) has the potential for use as a molecular recognition element in biosensors.