Use of a polyethylene glycol-peptide conjugate in a competition gel shift assay for screening potential antagonists of HIV-1 Tat protein binding to TAR RNA.
Interference of binding of Tat protein to TAR RNA in HIV-1-infected cells may be a useful therapeutic strategy for AIDS. An electrophoretic assay to screen potential low-molecular-weight (< 2 kDa) Tat antagonists has been established. A radiolabeled TAR RNA fragment (delta TAR) is retarded in mobility when bound by a Tat peptide-polyethylene glycol conjugate (Tat-PEG), which is used in place of the Tat protein. The assay determines the ability of a potential antagonist to compete with Tat-PEG for binding to delta TAR, as measured by interference with the gel shift of delta TAR. To discriminate between specific and nonspecific interactions, the assay is done in the absence or the presence of a 250-fold molar excess of tRNA.
Wang
J
J
Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey, USA.
Huang
S Y
SY
Choudhury
I
I
Leibowitz
M J
MJ
Stein
S
S
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001667:Binding, Competitive; D003864:Depression, Chemical; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D015696:Gene Products, tat / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D012331:RNA, Fungal / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D015533:Transcriptional Activation / Q000187:drug effects; D054322:tat Gene Products, Human Immunodeficiency Virus
D000998:Antiviral Agents; D015696:Gene Products, tat; D012331:RNA, Fungal; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D011092:Polyethylene Glycols; D012343:RNA, Transfer