Probing and regulating the activity of cellular enzymes by using DNA tetrahedron nanostructures† †Electronic supplementary information (ESI) available: Experimental section and supplementary tables and figures. See DOI: 10.1039/c9sc01912j

Given the essential role of apurinic/apyrimidinic endonuclease (APE1) in gene repair and cancer progression, we report a novel approach for probing and regulating cellular APE1 activity by using DNA tetrahedrons.

purified by HPLC (Sangon Co., Shanghai, China), their sequences are summarized in Table S1 and the use of these strands is explained in the denotation. APE1, lambda exonuclease (λ exo), deoxyribonuclease I (DNase I), exonuclease III (Exo III), exonuclease I (Exo I), T7 exonuclease (T7 Exo), T5 exonuclease (T5 Exo), and their corresponding buffers were obtained from NEB (Ipswich, MA). All chemicals were used as received without additional purification. DNase/RNase free deionized water from Tiangen Biotech Co. (Beijing, China) was used in all experiments.
Preparation and Characterization of the DNA Tetrahedrons. To prepare the DNA tetrahedrons, the oligonucleotides (final concentration of each strand 1 μM) were mixed in 1×TAE-Mg 2+ buffer (40 mM Tris, 20 mM acetic acid, 12.5 mM MgCl2, and 2 mM EDTA, pH 8). The above solution was denatured at 80℃ for 10 min followed by cooling to 4℃ in 30 min by using a PCR thermocycler. The nanostructures were purified by centrifugation at 12000 rpm for 10 min by ultrafiltration devices (Vivaspin, Sartorius, 30k, 0.5 mL). The nanostructures were re-suspended in the TAE-Mg 2+ buffer. The non-labeled DNA nanostructures were characterized by 12.5% native PAGE which was operated at room temperature for 4 h at a constant voltage of 120 V. The gel was subsequently stained with SYBR Gold. To characterize the tetrahedron by AFM, a drop of 2 μL DNA solution was spotted onto freshly cleaved mica surface, and kept for 10 seconds to achieve strong adsorption. The sample drop was then washed off by 30 μL TAE-Mg 2+ buffer, and dried by compressed air. DNA samples were imaged by tapping-mode AFM (FastScan, Bruker) with OTESPA-R3 tips. The tip-surface interaction was minimized by optimizing the scan set-point.
Characterization of the APE1 activity by fluorescence dequenching assay. All the in-vitro enzymatic reactions were carried out in 50 μL sealed PCR tubes. Once APE1 was added, fluorescence was recorded immediately in the FAM channel (ex: 480 nm, em: 510 nm) of a realtime PCR cycler (Rotor-Gene Q, QIAGEN, Germany) at 37 °C with a time interval of 5 s. 10 mM EDTA (final concentration) was used to terminate the reaction. The products were characterized by 12.5% native PAGE.
Cell culture and fluorescence imaging of the APE1 activity. A549, Hela and HEK-293 cell lines were cultured in 1640 medium supplemented with 1% Penn/Strep and 10% fetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air. The cells were S-3 transferred to a laser confocal culture dish for fluorescence imaging in an appropriate density. The cells were incubated with 100 nM of DNA tetrahedrons (labeled with Cy3 and BHQ2) and other reagents in a low-fluorescence culture media (FluoroBrite DMEM, Thermo Fisher). Highly inclined and laminated optical sheet (HILO) fluorescence microscopy was constructed using a Nikon inverted microscopy (ECLIPSE, Ti−U) equipped with a 100×magnification, 1.49 numerical aperture (NA) TIRFM objective (Nikon) and an EMCCD cameras (ixon 897, Andor). For HILO illumination, the laser of 520 nm was coupled into a single-mode fiber (Solamere Technologies).
The fiber optic cable that delivers laser light to the microscopy was secured into a fiber launch fitted with an XY fiber holder mounted atop a micrometer-driven optical rail for Z adjustment Bands on blots were visualized using enhanced chemiluminescence (ECL) detection system (5200, Tanon, China). α-tubulin and lamin b1 were used as reference for cytoplasmic protein and nuclear protein, respectively.