Monitoring helicase activity with molecular beacons

Abstract

A high-throughput, fluorescence-based helicase assay using molecular beacons is described. The assay is tested using the NS3 helicase encoded by the hepatitis C virus (HCV) and is shown to accurately monitor helicase action on both DNA and RNA. In the assay, a ssDNA oligonucleotide molecular beacon, featuring a fluorescent moiety attached to one end and a quencher attached to the other, is annealed to a second longer DNA or RNA oligonucleotide. Upon strand separation by a helicase and ATP, the beacon strand forms an intramolecular hairpin that brings the tethered fluorescent and quencher molecules into juxtaposition, quenching fluorescence. Unlike currently available real-time helicase assays, the molecular beacon-based helicase assay is irreversible. As such, it does not require the addition of extra DNA strands to prevent products from re-annealing. Several variants of the new assay are described and experimentally verified using both Cy3 and Cy5 beacons, including one based on a sequence from the HCV genome. The HCV genome-based molecular beacon helicase assay is used to demonstrate how such an assay can be used in high-throughput screens and to analyze HCV helicase inhibitors.

Figure 1. A molecular beacon based helicase assay

Comparison of Cy3 (A) and Cy5 (B) fluorescently labeled molecular beacons. Two substrates were designed with each beacon to evaluate the impact of duplex length and its ability to form hairpins. (C) Pseudo-first order rate constant describing fluorescence decay upon ATP addition (k_obs) versus duplex length. All reactions were performed in triplicate, and error bars represent standard deviation between independent reactions. Fluorescence obtained with substrate blanks (reactions without substrate) were subtracted from each reaction.

Figure 2. Ability of HCV Helicase to displace DNA and RNA bound molecular beacons

(A) Helicase substrates. (B) Fluorescence change upon helicase and ATP addition with the Cy3 beacon annealed to DNA or RNA. Reaction conditions were as described in Materials and Methods except that the substrates were present at 25 nM and enzyme at 125 nM. (C) Relationship between enzyme concentration and the rate constant describing fluorescence decay (k_obs) upon addition of ATP.

Figure 3. FRET based assay using molecular beacons

(A) Substrate. The signal caused by loss of FRET corresponds to the separation of the “donor” strand, a region of 19 base pairs. (B) Change in FRET in a helicase-substrate complex following ATP addition. The first order rate constant describing fluorescence decay, k_obs, is 0.23 for this reaction, very similar to that seen with the 19 bp substrate from Figure 1A. Fluorescence obtained with substrate blanks (reactions without substrate) are subtracted from each reaction.

Figure 4. A HCV genome-based molecular beacon assay

(A) Comparison of a molecular beacon assay to a conventional electrophoretic mobility shift (EMSA) gel-based helicase assay. Two simultaneous reactions were run with the substrate shown. One was monitored continuously for fluorescence (grey line), and aliquots were removed from the other and analyzed on three separate gels (squares). Fluorescence obtained from substrate blanks is subtracted and fractional fluorescence (F/F_o) is plotted. The error bars denote the standard deviation among the EMSAs and the dashed line represents the 95% confidence band for data derived from the EMSAs. (B) Sample gel from one of the EMSAs. Lane 1 represents an aliquot removed before adding ATP to start the reaction (t₀); lanes 2-10 represent aliquots removed after 20 s, 40 s, 1 min, 2 min, 3 min, 5 min, 7.5 min, 10 min, and 20 min. Lane 11 is the [³²P]oligonucleotide alone and lane 12 is substrate alone. (C) The unwinding reaction as a function of helicase concentration. (D) The unwinding reaction (25 nM helicase) as function of temperature. Because of the rapid progress of the reaction at elevated temperatures, this data was collected using a rapid-mixing stopped-flow device. (E) Substrate stability. Very little fluorescence change is observed for 12 hours when the substrate is incubated in reaction buffer without enzyme or ATP at 22 °C (black line). The inset shows annealing curves for the same substrate at 5 nM (black), 50 nM (red) and 500 nM (blue). Fluorescence was monitored while decreasing temperature from 95 °C.

Figure 5. High throughput screening

(A) Representative data from two continuously monitored inhibited and non-inhibited reactions. (B), (C) 104 inhibited and 103 non-inhibited reactions, carried out simultaneously in a 384 well plate with a final reaction volume of 50 μL. Identical sets of the same reactions were repeated on two different days. (B) Data obtained with a Cary Eclipse microplate reader with excitation/emission measured at 643/667 nm (slit widths of 5/10 nm, respectively) (C) Data obtained with black plates in a Tecan microplate reader with excitation/emission measured at 643/670 nm (9/20 nm slit widths). Substrate blanks (reactions without substrates) were not subtracted from reactions. (D) Data using the fluorescent assay to evaluate four inhibitors. dT₁₈ competes with the DNA substrate, while αβ-methylene-ATP and βγ-imido-ATP are non-hydrolysable ATP analogs that compete with ATP. NS3pep is a potent, newly described peptide inhibitor of NS3 helicase with the sequence RRGRTGRGRRGIYR (33).