Fluorescence Binding Assay of Atl1 Protein with the SIMA Labelled Oligonucleotide Containing the Tricyclic Nucleoside Analogue of O6-methyl-2'-deoxyguanosine

Abstract

The synthesis of phosphoramidite (DNA building block) containing the tricyclic nucleoside analogue of O6-methyl-2-deoxyguanosine was carried out and its Interactions with Atl proteins was quantified through fluorescent assay (fluorescent intensity monitoring). In fluorescent assay the interaction of DNA with protein is quantified by measuring the dissociation constant, KD. Usually the DNA is labelled with fluorophore (such as FAM, HEX or SIMA) which acts as a probe during the binding titration. Binding of protein to DNA substrate that is fluorescently labelled can cause changes in the fluorescent signal to that label since its interaction with protein changes its chemical environment from that in free solution. In this assay a series of fluorescent measurements were taken whilst increasing the amount of protein relative to the DNA. The binding curves were generated by plotting protein concentration against fluorescent intensity and the data were fitted by the non-linear least-squares regression using Kaleida Graph (I = Imax + [ (D+E+KD) – ( (D+E+KD)2 – (4DE) )0.5]    (Imin - Imax)/2D). The ODN containing the tricyclic analogue bound poorly with the Atl 1 protein with the KD = 194±29. The syntheses of the ODN with the modification at the O6-position as well as the florescence assay were reported here.