T4 polynucleotide kinase

EMSA was performed using a commercial assay kit (Cat NO. E3300, Gel Shift Assay System; Promega, Madison, WI, USA) to evaluate the NF-κB DNA-binding activity as previously reported. Briefly, the HeLa nuclear extract was used as a positive control. The NF-κB probe(5′-AGTTGAGGGGACTTTCCCAGGC-3′) was then mixed with [γ⁻³²P] ATP (Cat No. GS056; Beyotime, Shanghai, China) and T4-polynucleotide kinase (Cat NO. D7098L; Beyotime, Shanghai, China). The binding mixture was then added to the nuclear extract (40 μg protein) and incubated at 37 °C for 10 min. Then, the reaction mixture was subjected to electrophoretic separation on 4% native polyacrylamide gel in 0.5 × TBE buffer. Following the transmembrane procedure, visualization was conducted using HRP-labeled Streptavidin (Cat NO. A0303; Beyotime, Shanghai, China).

Plasmid pUC19, DH5α competent cells, Taq DNA polymerase, X-gal, and dNTPs were purchased from Tiangen Biotec Co., Ltd. (Beijing, China). Vent (exo^-) DNA polymerase and restriction enzymes (HindIII, EcoRI, and PstI) were obtained from New England Biolabs Inc (Ipswich, MA, USA). SYBR® gold nucleic acid gel stain was purchased from Invitrogen, Ltd. (Paisley, UK). T4 DNA ligase and T4 polynucleotide kinase were purchased from Beyotime (Shanghai, China). Plasmid small-extraction kit and 40% acrylamide (acrylamide:bis-acrylamide 19:1) were from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The E.Z.N.A.® Cycle-Pure Kit was obtained from Omega Biotek Inc (Georgia, USA). All oligonucleotides used in this study were purchased from Suzhou Beixin Biotechnology Co., Ltd. Ultrapure water was prepared using double distilled water (UPW, 18 MΩ·cm).
The following equipments were used: PAGE electrophoresis system (Junyi JY200C, Beijing, China), Micro high-speed centrifuge (Xiangyi TG16-W, Changsha, China), Microvolume UV–Vis spectrophotometer (NanoDrop2000 Thermo Fisher, Boston, Massachusetts, USA), ChemiDoc™ MP Imaging System (BioRad, Hercules, California, USA), MJ MiNi Thermal Cycler (Bio-Rad PTC-1148, Hercules, California, USA), and High-speed refrigerated centrifuge (Xiangyi TGL-16, Changsha, China).

The secondary structures of circMIB2 were predicted at [NaCl] = 1.0 M and 37 °C by the UNAfold Web Server. According to the prediction results, the optimal cyclization site of circMIB2 was determined, and the complete linear RNA of circMIB2 (L-circMIB2) was synthesized from this cyclization site. For ligation experiments, a phosphate was in advance introduced to the 5’-position of L-circMIB2 by using T4 polynucleotide kinase (Beyotime) and T4 RNA Ligase 2 (Beyotime) [28 (link)]. Finally, circMIB2 was purified by ethanol precipitation.