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

Abstract

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.

Highly sensitive detection of human thrombin in serum by affinity capillary electrophoresis/laser-induced fluorescence polarization using aptamers as probes

“…CE analysis of each sample was repeated three times. The fluorescence polarization (FP) values were calculated from the obtained intensity of the horizontally polarized fluorescence (I h ) and vertically polarized fluorescence (I v ) according to the following equation [34]:…”

“…It is also possible to differentiate the protein-DNA complexes with different binding stoichiometry by fluorescence polarization. Previous study demonstrated that the two complexes of human immunodeficiency virus type 1(HIV-1) reverse transcriptase (RT) and RT 26 aptamer with stoichiometry of 1:1 and 1:2 showed significantly different fluorescence polarization value (P = 0.098 for 1:1, and P = 0.039 for 1:2) [34]. In our case, since only one complex peak was observed, the complexes of thrombin-aptamer with different binding stoichiometries should be overlapped if they are to formed, and the fluorescence polarization of the complex peak should change with the varied concentration of thrombin.…”

Bioanalytical interaction studies executed by preincubation affinity capillary electrophoresis

“…In addition, using LIF polarization detection in pre-eq CZE studies it is possible to identify complex formation even in the absence of separation between the free fluorescent molecule and the complex [160]. In a recent study, the polarization detection was useful for identification of the stoichiometries of the formed complexes [142]. The sensitivity of LIF polarization was reported to be of the same magnitude as LIF detection [160].…”

“…Upon excitation with plane-polarized light the emitted fluorescence of a fluorescently labeled compound becomes depolarized due to the rotation of the molecule during excitation. Binding to another molecule, e.g., a protein will lower the extent of depolarization because the larger complex will rotate more slowly and the emitted light will be more polarized leading to higher polarization values [142,159]. Thus, the peaks related to the unbound fluorescent probe and the complex will have different polarization allowing identification of the complex.…”

“…Other examples related to the molecular biology of gene regulation are studies on DNA/oligonucleotide interactions with sea urchin embryo transcription factor [134], yeast transcriptional activator GCNK58 construct [138], MyoD and E47 transcription factors [139], histones H2B and H4 [136], ssDNA-binding protein [140], and serum response factor [141]. Fu et al [142] determined the stoichiometry of HIV-1 reverse transcriptase complexes with synthetic DNA aptamers originally designed as inhibitors of the polymerase site of HIV-1 reverse transcriptase using LIF detection in the pre-eq CZE format. RNA-peptide interactions were investigated by Mucha et al [143,144].…”