Use of the SYBR Green dye for measuring helicase activity

Label-free luminescence switch-on detection of hepatitis C virus NS3 helicase activity using a G-quadruplex-selective probe

“…Here, ciprofloxacin was chosen as a model inhibitor of helicase. 56 The luminescence signal of the system was diminished in the presence of ciprofloxacin in a dose-dependent manner, with a decrease of about 40% observed at a ciprofloxacin concentration of 10 μM ( Fig. 4c ).…”

“…The inhibitory effect of ciprofloxacin on helicase activity in our assay is comparable to that reported by Ali and co-workers. 56 Furthermore, ciprofloxacin has no direct quenching effect on the luminescence of 9 ( Fig. 4d ) or the 9 /G-quadruplex ensemble ( Fig.…”

“…54 , 55 Ali and co-workers used SYBR Green dye, which is strongly fluorescent in the presence of dsDNA but not ssDNA, for the detection of helicase activity. 56 A similar principle was utilized by Kowalczykowski and co-workers to construct a switch-off platform for helicase activity using ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 or thiazole orange. The groups of Frick and Boguszewska-Chachulska reported an approach for monitoring helicase activity using molecular beacons.…”

Simian Virus 40 Large T Antigen as a Model to Test the Efficacy of Flouroquinolones against Viral Helicases

“…Purified SV40 LT-Ag was purchased from CHIMERx, USA. As described previously [ 9 ], the helicase substrate was prepared by mixing single strands of complimentary oligonucleotides in 1:1 ratio to a final concentration of 20 μM each, heated to 95°C for 5 min, and then allowed to anneal by gradual cooling to room temperature for 1 hr. Typically, each helicase reaction contained 20μl 5x helicase buffer (200mM Tris pH-7.6, 25mM MgCl2, 20mM DTT, 125mM KCl, 10% glycerol and 0.5μg/ulBSA), 1mM ATP, 10μM DNA oligo substrate, 1x SYBR Green I and 1.44μM purified helicase, in a total volume of 100μl.…”

“…The LT-Ag has served as a model helicase also for the screening of antiviral drugs that target viral helicases [ 8 ]. In our previous study, LT-Ag was used as a model helicase to develop a non-radioactive helicase assay [ 9 ]. For the current study, using in vitro and in silico methods, we screened a batch of 18 fluoroquinolones to assess their potential as antivirals by virtue of their binding to, and inhibition of, the LT-Ag helicase.…”