Construction of a Dendritic Nanoassembly-Based Fluorescent Biosensor for Electrostatic Interaction-Independent and Label-Free Measurement of Human Poly(ADP-ribose) Polymerase 1 in Lung Tissues

Poly(ADP-ribose) polymerase 1 (PARP-1)is responsiblefor catalyzingthe creation of poly(ADP-ribose) polymer and involved in DNA replicationand repair. Sensitive measurement of PARP-1 is critical for clinicaldiagnosis. However, the conventional electrostatic attraction-basedPAPR-1 assays usually involve laborious procedures, poor sensitivity,and false positives. Herein, we demonstrate the construction of adendritic nanoassembly-based fluorescent biosensor for electrostaticinteraction-independent and label-free measurement of human PARP-1in lung tumor tissues. When PARP-1 is present, the specific double-strandedDNA (dsDNA)-activated PARP-1 transfers the ADP-ribosyl group fromnicotinamide adenine dinucleotide (NAD(+))/biotinylated NAD(+) to the PARP-1 itself, resulting in the formation of biotinylateddsDNA-PARP-1-PAR polymer bioconjugates that can be captured by magneticbeads. Upon the addition of TdT, APE1, and NH2-modifiedT-rich probe, the captured dsDNAs with dual 3 & PRIME;-OH termini initiateTdT-activated APE1-mediated hyperbranched amplification to produceabundant dendritic DNA nanoassemblies that can be stained by SYBRGreen I to generate a high fluorescence signal. This biosensor ischaracterized by a template-free, electrostatic interaction-independent,high sensitivity, and label-free assay. It enables rapid (less than3 h) measurement of PARP-1 with a limit of detection of 4.37 x10(-8) U/& mu;L and accurate measurement of cellularPARP-1 activity with single-cell sensitivity. Moreover, it is capableof screening potential inhibitors and discriminating the PARP-1 levelin normal person tissues and lung cancer patient tissues, with greatpotential in PARP-1-related clinical diagnosis and drug discovery.

Automated summary

A dendritic nanoassembly-based fluorescent biosensor is developed for the measurement of PARP-1 in lung tumor tissues. This label-free assay is template-free and electrostatic interaction-independent, with high sensitivity and rapid detection time. It has great potential in PARP-1-related clinical diagnosis and drug discovery.