Study of binding stoichiometries of the human immunodeficiency virus type 1 reverse transcriptase by capillary electrophoresis and laser-induced fluorescence polarization using aptamers as probes.
Binding stoichiometries between four DNA aptamers (RT12, RT26, RTlt49, and ODN93) and the reverse transcriptase (RT) of the type 1 human immunodeficiency virus (HIV-1) were studied using affinity CE (ACE) coupled with LIF polarization and fluorescence polarization (FP). The ACE/LIF study showed evidence of two binding stoichiometries between the HIV-1 RT protein and aptamers RT12, RT26, and ODN93, suggesting that these aptamers can bind to both the p66 and p51 subunits of the HIV-1 RT. Only one binding stoichiometry for aptamer RTlt49 was found. The affinity complexes were easily separated from the unbound aptamers; however, the different stoichiometries were not well resolved. A complementary technique, FP, was able to provide additional information about the binding and supporting evidence for the ACE/LIF results. The ACE/LIFP study also revealed that the FP values of the 1:1 complexes of the HIV-1 RT protein with aptamers RT12, RT26, and ODN93 were always much greater than those of the 1:2 complexes. This was initially surprising because the larger molecular size of the 1:2 complexes was expected to result in higher FP values than the corresponding 1:1 complexes. This phenomenon was probably a result of fluorescence resonance energy transfer between the two fluorescent molecules bound to the HIV-1 RT protein.
Fu
Hao
H
Environmental Health Sciences, Department of Public Health Sciences, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
Guthrie
Jeffrey W
JW
Le
X Chris
XC
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D006801:Humans; D007834:Lasers; D011485:Protein Binding
D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D054303:HIV Reverse Transcriptase