Nb mva1269i

T4 DNA ligase and phi29 polymerase (10 units per μL) were obtained from New England Biolabs (Beijing, China). Nb.Mva1269I (10 units per μL) was bought from Fermentas. Deoxyribonucleoside triphosphates (dNTPs, 100 mM), NaCl and KCl were bought from Beijing DingGuo Biotech. Co., Ltd. All solutions were prepared using ultrapure water. The DNA sequences were purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. The molecular beacon (MB) modified with 5′-FAM and 3′-dabcyl was purified through high-performance liquid chromatography (HPLC). Other DNA sequences without modification were purified through denaturing polyacrylamide gel electrophoresis (PAGE). The sequences of the oligonucleotide probes used in this work are listed in Table S1 and Fig. S1.† Five single-stranded oligonucleotides (H1, H2, H3, H4, H5) were hybridized with each other to form the track with three protruding single-stranded branches (A, B, C). The sequences of the RCA products of C1 and C2 are complementary to the underlined sequences of the MB.

All enzymes were from New England Biolabs, with the exception of wheat germ topoisomerase I (Promega in early experiments, later Inspiralis), calf thymus topoisomerase I (Invitrogen), and nicking endonuclease Nb.Mva1269 I (Fermentas), which was preferred to its isoschizomer Nb.BsmI for its optimal digestion temperature of 37°C, as opposed to 65°C for Nb.BsmI.
Note: care was taken not to overdigest with Nb.BtsI, as this nicking enzyme tended to show non-specific star activity when used at high concentration (data not shown).