Surface plasmon resonance study on HIV-1 integrase strand transfer activity
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Understanding the molecular mechanism of HIV-1 integrase (IN) activity is critical to find functional inhibitors for an effective AIDS therapy. A robust, fast, and sensitive method for studying IN activity is required. In this work, an assay for real-time label-free monitoring of the IN activity based on surface plasmon resonance was developed. This assay enabled direct monitoring of the integration of a viral doubled-stranded (ds) DNA into the host genome. The strand transfer reaction was detected by using two different DNA targets: supercoiled plasmid (pUC 19) and short palindrome oligonucleotide. The effect of the length of the DNA target on the possibility to monitor the actual process of the strand transfer reaction is discussed. The surface density of integrated ds-DNA was determined. IN binding to the oligonucleotide complexes and model DNA triplexes in the presence of various divalent ions as metal cofactors was investigated as well. The assay developed can serve as an important analytical tool to search for potential strand transfer reaction inhibitors as well as for the study of compounds interfering with the binding of ds long terminal repeats–IN complexes with the host DNA.
KeywordsSurface plasmon resonance Biosensor HIV-1 integrase Strand transfer reaction
The authors would like to thank Kamila Moquin and Praskovia Boltovets for help with the experiments and Sergey Leshkov for deposition of thin films for SPR chips. This research was supported by the Czech National Science Foundation (202/05/0628) and the Academy of Sciences of the Czech Republic (AV0Z20670512, 203/05/P557, and KAN200670701).