Specificity of DNA triple helix formation analyzed by a FRET assay
© Reither and Jeltsch; licensee BioMed Central Ltd. 2002
Received: 28 August 2002
Accepted: 12 September 2002
Published: 12 September 2002
A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis.
We have analyzed triplex formation of two polypurine triplex forming oligodeoxynucleotides (TFOs) using fluorescence resonance energy transfer (FRET). Under our conditions, the TFOs bind to their cognate double strand DNAs with binding constants of 2.6 × 105 and 2.3 × 106 M-1. Our data confirm that the polypurine TFO binds in an antiparallel orientation with respect to the polypurine DNA strand and that triplex formation requires Mg2+ions whereas it is inhibited by K+ions. The rate of formation of triple helices is slow with bimolecular rate constants of 5.6 × 104 and 8.1 × 104 min-1 M-1. Triplex dissociation was not detectable over at least 30 hours. Triplex formation is sequence specific; alteration of a single base pair within the 13 base pairs long TFOs prevents detectable triplex formation.
We have applied a FRET assay to investigate the specificity of DNA triple helix formation. This assay is homogeneous, continuous and specific, because the appearance of the FRET signal is directly correlated to triplex formation. We show that polypurine TFOs bind highly specifically to polypurine stretches in double stranded DNA. This is a prerequisite for biotechnical applications of triple helices to mediate sequence specific recognition of DNA.
It has been discovered in 1957 that a homopyrimidine DNA strand (triplex forming oligonucleotide, TFO) can bind to a homopurine/homopyrimide DNA duplex in the major groove by forming Hoogsteen base pairs with the homopurine strand . The Hoogsteen base pairing scheme mediates sequence specific recognition of the double stranded DNA by the TFO where an AT base pair is recognized by a T and a GC base pair by a C that is protonated at N3 (reviews: [2–4]). Later it was found that homopurine strands can also specifically form a DNA triplex in which the AT base pair is contacted by an A and the GC base pair by a G . Triple helix formation with purine rich TFOs is less pH sensitive but requires divalent cations like Mg2+[6–8]. In either case, the two pyrimidine strands and the two purine strands must be arranged in an antiparallel orientation to form a stable triplex (reviews: [2–4]). Triple helix formation has been employed for various purposes in biotechnology like gene targeting, mutagenesis and inhibition of gene activity (reviews: [9–11]).
Results and Discussion
Detection of triple helix formation by FRET
We have investigated the biochemical and biophysical properties of a stable purine(purine-pyrimidine) triplex , and checked for its sequence specificity. We used a 21 mer double stranded DNA, DS1, that contains a homopurine stretch of 16 base pairs (Fig. 1). The pyrimidine strand carries a 6-carboxyfluorescein (FAM) label on its 5' end. The corresponding 13 mer TFO1 is 5' labeled with 5-carboxytetramethylrhodamine (TAMRA). FAM and TAMRA form a FRET pair with an R0 of 49–54 Å . After triplex formation, the donor and acceptor groups are separated by 4 base pairs which according to computer modeling corresponds to a distance of approximately 15 Å. Therefore, a direct interaction of the fluorophors, which is unfavorable for the assay, cannot occur, but the probes are well within a distance that should lead to highly efficient FRET.
To check the general feasibility of this technique, we have used an additional pair of DNA and TFO. To this end, the sequence of TFO1 was changed at four positions to create TFO2 that should specifically bind to DS2. Triple helix formation of TFO2 and DS2 has not been studied so far. As shown in Fig. 2B, the results obtained with the TFO2/DS2 pair are very similar to the TFO1/DS1 pair. This finding demonstrates that triplex formation with polypurine TFOs is a general phenomenon not restricted to certain sequences.
Determination of the biophysical properties of the different triple helices
The total efficiency of the FRET process can be estimated at the highest concentration of the TFO where the DNA is almost saturated with TFO. Under these conditions, the donor fluorescence is quenched by more than 85% (Fig. 4). This result confirms that the TFO binds to the DNA in an antiparallel orientation with respect to the polypurine strand [5, 25], because in a parallel orientation the distance between the donor and acceptor groups would not allow highly efficient FRET.
For the dissociation reactions, triplex formation was performed by annealing at 55°C in a small volume of buffer. Then, the triplex was diluted into a solution containing a large excess of TFO not carrying the TAMRA label. Fluorescence was determined over 30 hours, but no significant change in the FRET efficiency was observed, indicating that dissociation occurs very slowly under these conditions. In contrast, rapid dissociation of the triplex was observed after addition of 20 mM EDTA to the binding buffer confirming that the experimental setup was suited to detect triplex dissociation (data not shown).
Sequence specificity of triplex formation
In this work we have investigated formation and specificity of DNA triplexes using a FRET assay. This assay has many important advantages over alternative assay systems to monitor triplex formation: 1) It is very fast, convenient and requires only small amounts of sample. 2) It is a direct assay, because only triplex formation can lead to the specific FRET signal. 3) It is homophasic and does not depend on the separation of free and bound TFO, which always tends to shift the equilibrium. 4) It allows continuous measurements and thus is suited to follow kinetics of triplex formation and dissociation. We show that triplex formation is highly specific, because exchange of one base pair in the double stranded DNA or in the TFO prevents triplex formation. This high specificity is a prerequisite for the usage of triple helices in biotechnology, e.g. for gene targeting, reduction of gene expression or mutagenesis.
Purified oligodeoxynucleotides were purchased from MWG (Eberberg, Germany). The homogeneity of the oligonucleotides was checked on denaturing polyacrylamide gels. Triplex formation was assayed in binding buffer (20 mM HEPES pH 7.5, 50 mM Na-acetate, 10 mM MgCl2). For most experiments, 500 nM double stranded DNA and 2500 nM TFO were mixed, annealed by heating to 55°C for 10 min and equilibrated at 37°C. Fluorescence was measured using a Hitachi F4500 spectrofluorometer using 50 μl fluorescence cuvettes. Excitation was at 470 nm, emission was determined between 480 and 690 nm. Excitation and emission slits were at 5 and 2.5 mm, respectively. Spectra were recorded at a scanning speed of 2400 nm/min, usually 9–16 spectra were averaged to improve the signal to noise ratio. To calculate the efficiency of FRET, we used the fluorescence of the acceptor group (TAMRA) which was obtained by averaging the fluorescence emission spectra between 576 and 584 nm. To measure the binding constant of the TFOs to their corresponding double stranded DNA increasing amounts of TFO (10 nM – 10 μM) were added to a constant amount of DNA (500 nM). The samples were incubated for 10 hours at 37°C and the fluorescence analyzed. To determine the equilibrium binding constants, data were fitted as described . Association kinetics were determined using three different concentrations of TFO (1, 2.5 and 4 μM) and a constant amount of double stranded DNA (500 nM). TFO and DNA were mixed, immediately placed in the spectrofluorometer and temperature maintained at 37°C. Fluorescence was scanned at defined time points for 30 min. To determine the rate constant of association all data sets were globally fitted to a bimolecular association reaction as described .
Thanks are due to A. Pingoud, H. Gowher and K. Eisenschmidt for discussions and B. Kleiber for technical assistance. This work has been supported by the BMBF Biofuture program and the European Union.
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