Abstract
This work reports the development of a fluorescence method for the detection of poly(ADP-ribose) polymerase-1 (PARP1), in which a phenylboronic acid-modified fluorescein isothiocyanate dye (FITC-PBA) was used to recognize the formed poly(ADP-ribose) (PAR) polymer. The detection system was designed by conjugating recombinant streptavidin (rSA) with PARP1-specific double-stranded DNA (dsDNA) through streptavidin-biotin interaction. Capture of PARP1 via rSA-biotin-dsDNA allowed for the poly-ADP-ribosylation (PARylation) of both rSA and PARP1 in a homogeneous solution. The resulting rSA-biotin-dsDNA/PAR conjugates were then captured and separated via the commercialized nitrilotriacetic acid-nickel ion-modified magnetic bead (MB-NTA-Ni) through the interaction between NTA-Ni on MB surface and oligohistidine (His(6)) tag in rSA. The PAR polymer could capture the dye of FITC-PBA through the borate ester interaction between the boronic acid moiety in PBA and the cis-diol group in ribose, thus causing a decrease in fluorescence signal. The PARylation of streptavidin and the influence of steric hindrance on PARylation efficiency were confirmed using reasonable detection strategies. The method showed a wide linear range (0.01~20 U) and a low detection limit (0.01 U). This work should be valuable for the development of novel biosensors for the detection of poly(ADP-ribose) polymerases and diol-containing species.