A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity

Abstract

We have devised a simple high-throughput screening compatible fluorescence polarization-based assay that can be used to detect the elongation activity of nucleic acid polymerase enzymes. The assay uses a 5' end-labeled template strand and relies on an increase in the polarization signal from the fluorescent label as it is drawn in toward the active site by the action of the enzyme. If the oligonucleotide is sufficiently short, the fluorescence polarization signal can also be used to detect binding prior to elongation activity. We refer to the nucleic acid substrate as a polymerase elongation template element (PETE) and demonstrate the utility of this PETE assay in a microtiter plate format using the RNA-dependent RNA polymerase from poliovirus to extend a self-priming hairpin RNA. The PETE assay provides an efficient method for screening compounds that may inhibit the nucleic acid binding or elongation activities of polymerases.

Figure 1

Schematic illustration of the RNA oligonucleotides used to measure RNA binding and elongation by the poliovirus polymerase with fluorescence polarization. The hairpin structures are followed by a single stranded template sequence that is labeled with a fluorescein molecule at its 5’ end. The addition of NTPs leads to elongation and translocation of the template RNA and the fluorescein label eventually becomes immobilized as it contacts the protein surface, increasing its FP signal in an enzyme activity dependent manner.

Figure 2

A) Binding of an “8-6” RNA hairpin to poliovirus polymerase as detected by fluorescence polarization of a fluorescein label on the 5′ end of the RNA. Experiments were carried out using 10 nM RNA and the dissociation constants resulting from fitting the data to a binding isotherm are listed in Table 1. B) Gel electrophoresis analysis showing 3D^pol dependent single nucleotide elongation steps of a ³²P 5′ end labeled RNA hairpin with a _3′CAG_5′ templating sequence. In the presence of enzyme the RNA is elongated stepwise from a 26mer to a 29mer by the incorporation of G, U, and C, and the last lane shows that the 2^nd and 3^rd nucleotides are not added if the first nucleotide triphosphate (GTP) is omitted from the reaction.

Figure 3

Stepwise elongation of the hairpin primer-template RNAs monitored by fluorescence polarization. The RNAs were pre-incubated with 3D^pol and ATP at 4°C for 30 minutes prior to being warmed up to room temperature for the FP assays. UTP was then added, triggering the addition of 2, 4, or 6 uracils depending on the length of the template sequence (Table 1) and stalling the 3D^pol-RNA complexes three nucleotides from the 5′ end of the template strand. The RNA was then fully elongated by the stepwise addition of the remaining NTPs. Note that regardless of the RNA length, only the incorporation of the penultimate nucleotide leads to a significant increase in the FP signal. We attribute this to an immobilization of the fluorescein probe on the protein surface as the 5′ end of the templating strand is translocated into the active site.

Figure 4

A) Kinetic analysis of cognate and non-cognate base incorporation by 3D^pol. Polymerase was pre-incubated with the “8-4” RNA hairpin and the 1^st, 3^rd, and 4^th required NTPs at concentrations of 0.2 μM each. This results in a stable complex that has incorporated the first base (A) and is poised for the addition of the missing second nucleotide (C) before fully elongating by incorporating U and G. Addition of the cognate CTP at 0.2 μM concentration results in complete elongation at a rate too rapid to detect by the microplate reader. The incorporation of non-cognate uracil can be driven by increasing the UTP concentration one thousand-fold to 200 μM, resulting in a time dependent exponential increase in the FP signal, while addition of the bulkier ATP or GTP does not drive mis-incorporation. B) Plot showing that the uracil incorporation rate constant is linear with respect to UTP concentration. The rates were obtained by fitting the temporal uracil incorporation curves (e.g. panel A) to a single exponential function. C) Data showing an absence of an increase in the FP signal when the polymerase is inhibited by adding EDTA to chelate the Mg²⁺ ions required for elongation activity.