A novel RNA motif that binds efficiently and specifically to the Ttat protein of HIV and inhibits the trans-activation by Tat of transcription in vitro and in vivo.
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 RNATat, 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 RNATat, including binding kinetics, identification of functional groups for Tat binding, and inhibition of Tat function.


RESULTS
The binding affinity of the isolated aptamer RNATat to Tat-1 was 133 times higher than that of authentic TAR-1 RNA. RNATat is composed of inverted repeats of two TAR-like motifs, and even though RNATat had two Tat-binding core elements, the interaction with Tat took place at a molar ratio of 1 : 1. Several functional groups of aptamer RNATat responsible for Tat binding were identified. The selected aptamer RNATat 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, RNATat, retained strong affinity for Tat even in the presence of a large excess of HIV TAR. RNATat binds efficiently to Tat proteins or peptides derived from either HIV-1 or HIV-2. Unlike TAR RNA, RNATat affinity does not depend upon cellular proteins such as cyclin T1, thus RNATat has the potential for use as a molecular recognition element in biosensors.
Yamamoto
R
R
National Institute of Bioscience and Human Technology, University of Tsukuba, Japan.
Katahira
M
M
Nishikawa
S
S
Baba
T
T
Taira
K
K
Kumar
P K
PK
D001483:Base Sequence; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D017421:Sequence Analysis; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA