链霉亲和素偶联 DNA 用于硼酸亲和的多聚（ADP-核糖）聚合酶-1 的高灵敏度检测。
Streptavidin-Conjugated DNA for the Boronate Affinity-Based Detection of Poly(ADP-Ribose) Polymerase-1 with Improved Sensitivity.
机构信息
College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang 455000, China.
College of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, China.
出版信息
Biosensors (Basel). 2023 Jul 10;13(7):723. doi: 10.3390/bios13070723.
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) 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 -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.
这项工作报道了一种用于检测聚 ADP-核糖聚合酶 1（PARP1）的荧光方法的发展，其中使用了苯硼酸修饰的异硫氰酸荧光素染料（FITC-PBA）来识别形成的聚 ADP-核糖（PAR）聚合物。该检测系统通过将重组链霉亲和素（rSA）与 PARP1 特异性双链 DNA（dsDNA）通过链霉亲和素-生物素相互作用进行偶联来设计。通过 rSA-生物素-dsDNA 捕获 PARP1，允许在均相溶液中对 rSA 和 PARP1 进行多聚 ADP-核糖基化（PARylation）。然后，通过商业化的氮三乙酸-镍离子修饰的磁性珠（MB-NTA-Ni）通过 MB 表面上的 NTA-Ni 与 rSA 中的寡组氨酸（His）标记之间的相互作用，将形成的 rSA-生物素-dsDNA/PAR 缀合物捕获和分离。PAR 聚合物可以通过 PBA 中的硼酸部分与核糖中的 -二醇基团之间的硼酸酯相互作用捕获 FITC-PBA 的染料，从而导致荧光信号降低。通过合理的检测策略证实了链霉亲和素的 PARylation 以及空间位阻对 PARylation 效率的影响。该方法显示出较宽的线性范围（0.01~20 U）和较低的检测限（0.01 U）。这项工作对于开发用于检测聚 ADP-核糖聚合酶和含二醇物质的新型生物传感器应该是有价值的。
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