Journal: Nucleic Acids Research
Article Title: Specific binding of a hexanucleotide to HIV-1 reverse transcriptase: a novel class of bioactive molecules
Figure Lengend Snippet: Binding of hexanucleotides to HIV-1 RT. ( A ) Relative binding of DNA and homologous RNA hexamers as well as a RT-directed aptamer to RT (5 μM). The bars indicate mean values and SD of representative experiments. Pairs of DNA and RNA versions of hexamers are indicated by blue color. *The aptamer sequences are: DNA aptamer, d(ATCCGCCTGATTAGCGATACTCAGAAGGATAAACTGTCCAGAACTTGGA), RNA aptamer, GGGAGAUUCCGUUUUCAGUCGGGAAAAACUGAA. ( B ) Titration of HIV-1 RT with radiolabeled Hex-S3 R (1 nM) applying a filter binding assay. The curve shows the best fit of the experimental data to a quadratic equation and yields a K d of 5.3 (±0.5) μM. ( C ) Mutational analysis of the importance of each nucleotide position of Hex-S3 for binding to RT. An adenosine was introduced at each of the positions. ( D ) Site-specific binding of Hex-S3 R modified by a 4-thio-uridine at position 4 to RT as shown by UV cross-linking. Left panel, Coomassie blue-stained gel; middle panel, autoradiograph; right panel, overlay. ( E ) The 3D model of the binding site of RT for Hex-S3 based on docking studies and the constraints explained in the text. Color code: yellow, Hex-S3; green, CNBr fragment (42–183 amino acid); red, CNBr fragment (231–356 amino acid); blue, remaining portions of the large subunit p66; gray, small subunit p51. ( F ) Protein target specificity of Hex-S3.
Article Snippet: For this matter, filter binding assays were performed with (i) the small subunit p51 of HIV-1 RT, (ii) HIV-2 RT, which is highly homologous to the HIV-1 RT (60% amino acid identity plus 13% amino acid homology), (iii) EIAV RT, (iv) T7 RNA polymerase and (v) BSA ( ).
Techniques: Binding Assay, Titration, Filter-binding Assay, Modification, Staining, Autoradiography