Assaying the activity of HIV-1 integrase with DNA-coated plates.
Integration of reverse transcribed viral DNA of HIV into host chromosomes is mediated by the viral enzyme, integrase. This enzymatic activity can be monitored in vitro by integration of a small labeled DNA (donor) into a second unlabeled DNA (target). The methodology usually involves isotope labeling and gel electrophoresis. To simplify the measurement, a method mimicking enzyme-linked immunosorbent assay (ELISA) procedures was developed. Fragments of DNA were adsorbed directly on 96-well plates and used as the target DNA. The donor was a synthetic 21-bp DNA duplex of HIV-1 U5 LTR; biotin was incorporated into the 5' end of one strand whose two nucleotides at the 3' end were specifically removed during the integration. As a result of integration, the biotin-labeled donor DNA was joined with the target DNA and became immobilized on plates. These integration products were then measured by binding of avidin-alkaline phosphatase on plates. The method is simple and straightforward and can easily be adapted for high throughput screening of integrase inhibitors.
Chang
Y C
YC
Institute of Microbiology and Immunology, National Yang-Ming University, Taiwan, ROC.
Ching
T T
TT
Syu
W J
WJ
D004247:DNA / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019427:HIV Integrase / Q000032:analysis* / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
D011993:Recombinant Fusion Proteins; D004247:DNA; D019427:HIV Integrase