An in silico and in vitro pipeline for the rapid screening of helicase modulators

To evaluate the potency of potential helicase modulators, we developed an assay of helicase enzyme activity. Using a DNA or RNA biotin labelled oligonucleotide and after the addition of a recombinant helicase, the nucleic acid unwinds, causing the emission of luminescence, which is quantified with a particular antibody. In our assay, one of the DNA oligos was biotinylated, while the other was labelled with digoxygenin (DIG), both in their 5’ termini. The biotin molecule immobilises the DNA duplex on a neutravidin-coated plate and the helicase activity is measured through the unwinding of DNA, due to ATP activation. The subsequent release of DIG-labelled oligos results in a luminescence signal measured with a chemiluminescence antibody. Our goal was to provide a high throughput screening method for potential helicase inhibitors. The method described in this paper has been demonstrated to be fast, easy and reproducible and doesn’t use radiochemicals.


Introduction
Helicase activity assays include analysis of ATPase activity, but it was shown that measuring the helicase unwinding activity is the best method for evaluating modulators of this class of enzyme (Borowski et al., 2002). This type of assay depends on the ability of the enzyme to separate the release strand of DNA or RNA from the template strand. Other methods measure the deposition of radio-labelled release strands after gel electrophoresis, thinlayered chromatography or scintillation counting (Borowski et al., 2001; Bartelma and Padmanabhan 2002;Alaoui-Ismaili et al., 2000). These methods could be enhanced via high-throughput screening, although the radioactive materials would be a problem. Another method detects DIG-labelled release strands by ELISA (Hsu et al., 1998). We propose a combination of the methods as mentioned above without radio-labelled molecules that can detect the residual release strand with a chemiluminescent antibody, giving a robust helicase assay and a stable readout, well suited to high-throughput screening.
Our goal was to provide a high throughput screening method for potential helicase inhibitors, and that is why we developed this fast, easy and reproducible assay, without using radiochemicals. The high reproducibility of the assay is obvious after the observation of only insignificant variations on a single 96-reaction plate. Helicase from the Hepatitis C Virus (HCV) was expressed and isolated through recombinant protein methods and later used in our assay.

Materials
All solutions were prepared using ultrapure water obtained by purifying deionised water. All reagents were stored at room temperature unless indicated otherwise.

1.
Escherichia coli induction: Prepare 4 conical flasks with 750 mL of LB and add 34 μg/mL chloramphenicol and 25 μg/mL kanamycin to each;

Methods
All procedures have to be carried out at room temperature unless specified otherwise.

1.
Insert the full-length HCV helicase coding region in a pET28a vector, with a Nterminal 6xHis-Tag region;

2.
verify the intactness of the gene before inducing the protein production;

3.
transform Escherichia coli cells (strain C41, DE3) with the helicase plasmid and inoculate the prepared LB flasks with them;

4.
induce the recombinant helicase production, adding 0.5 mM IPTG to each flask and then allow the cultures to grow for 3 hours at 18°C;

5.
resuspend the cell pellet from the 4 cultures in 30 mL Lysis buffer and then add lysozyme 100μg/mL and Triton X-100 0.1%;

6.
incubate on ice for 30 min and then sonicate four times for 20 sec with 15 sec intervals;

7.
centrifuge the suspension at 15.000 x g for 20 min;

8.
adjust the clarified homogenates to 10 mM imidazole and filter them through a 0.45 μm membrane;

10.
wash each column with five times the column volume of buffer S, containing 10 mM imidazole;

12.
immediately after the elution, exchange the buffer in the helicase-containing fractions for the Exchange buffer, via dialysis; this step is critical in order to avoid precipitation;

13.
evaluate the protein concentration using the Bradford assay with BSA as standard;

14.
create aliquots of the NS3 helicase and store at −80°C.
This recombinant protein preparation is estimated to be more than 85% pure by SDS gel electrophoresis and Coomassie blue staining, yielding almost 1.6 mg of HCV NS3 per 3 litres of E. coli cultures ( Figure 1).
Chemiluminesence readings were taken using all different control combinations (presence/ absence of helicase, DNA substrate and ATP) of the experiment to ensure the reliability of the measurements (Table 1).
We demonstrated that the NS3-mediated unwinding is proportional to the amount of DNA substrate in the well, but also to the HCV helicase concentration in the reaction. The reactions were ATP-dependent (Table 2 and Figure 2).

1.
Prepare for annealing by heating the oligonucleotide mix at 100°C for 5 min;

2.
incubate at 65°C for 30 min and then at 22° for 4 h, to allow gradual annealing;
Neutravidin coating of the 96-well plates

1.
Coat each of the 96 wells overnight at 4°C with 100 μl/well of a 5 μg/ml neutravidin solution in 0.5 M sodium carbonate buffer pH 9.3;

2.
wash the plates three times with 100μl/well of PBS and air-dry at room temperature.
Blocking with BSA

2.
incubate at 22°C for 2 h; by DIG labelling of the release strand. Other sequences could work, but a 3' single stranded region in the substrate is necessary to initiate the strand displacement (Tai et al., 1996).