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Nucleic Acids Research Pages 1802-1809  

5-(1-propargylamino)-2[prime]-deoxyuridine (UP): a novel thymidine analogue for generating DNA triplexes with increased stability
Introduction
Materials And Methods
   Chemical synthesis
   DNA oligonucleotide synthesis
   Chemicals and enzymes
   UV melting
   DNA fragments
   DNase I footprinting
   Gel electrophoresis
   Quantitative footprinting analysis
Results
   UV-melting
   Footprinting
Discussion
Acknowledgement
References

5-(1-propargylamino)-2[prime]-deoxyuridine (U<sup>P</sup>): a novel thymidine analogue for generating DNA triplexes with increased stability

5-(1-propargylamino)-2[prime]-deoxyuridine (UP): a novel thymidine analogue for generating DNA triplexes with increased stability

Jeevan Bijapur, Melanie D. Keppler1, Simon Bergqvist, Tom Brown and Keith R. Fox1,*

Department of Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ, UK and 1Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK

Received January 28, 1999; Revised and Accepted March 3, 1999

ABSTRACT

We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (UP). The intra-molecular triplex A6-L-T6-L-(UP)5T (L = two octanediol residues) shows a single UV-melting transition which is >20° higher than that of the parent triplex A6-L-T6-L-T6 at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by UP, in a pH dependent fashion. Triplex stability increases by ~12 K for each T to UP substitution. Quantitative footprinting studies have examined the interaction of three UP-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5[prime]-AGGAAGAGAAAAAAGAA. At pH 5.0, the UP-containing oligo-nucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (UP)6CUPT binds ~1000-fold more tightly than the unmodified oligonucleotide T6CTT. Oligonucleotides containing fewer UP residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.

INTRODUCTION

DNA triple helices, which were first demonstrated in 1957 (1), are formed by the addition of an oligonucleotide to the major groove of duplex DNA. Binding of the third strand is sequence-specific and is stabilised by the formation of hydrogen bonds to substituents on the duplex purine strand (2-5). Since these structures form in a sequence-specific fashion they have attracted considerable interest as agents which have the potential to inhibit gene expression in a sequence-specific fashion (6-9).

Two types of triplex have been described, which differ in the orientation of the third strand. Pyrimidine-rich oligonucleotides bind parallel to the duplex purine strand and are characterised by the formation of T·AT and C+·GC triplets (2,3,10), while purine-rich oligonucleotides bind in an antiparallel orientation forming G·GC, A·AT and T·AT triplets (11-14). Formation of the C+·GC triplet requires conditions of low pH (<6.0), necessary for protonation of the third strand cytosine. The free nucleoside has a pK of ~4.5, though this is elevated at isolated cytosines within triplex forming oligonucleotides, depending on their number and location (15). Several cytosine analogues have been synthesised in attempts to overcome this restriction (16-25).

A further limitation to the formation of triplexes is that they are less stable than corresponding duplexes as a result of charge repulsion between the three negatively charged phosphodiester backbones. For this reason triplex formation usually requires the presence of divalent metal ions (26). However, it has recently become apparent that isolated C+·GC triplets confer a much greater triplex stability than T·AT (15,27,28). This may be due to favourable interactions between the protonated cytosine ring and the stacked [pi]-electrons, or because the positive charge on the base partially neutralises the negative charge on the phosphodiester backbones. This discovery led us to suggest that the strength of the T·AT triplet might be increased by synthesising positively charged thymidine analogues. This approach to neutralising the charge repulsion has previously been attempted by modifying the phosphodiester backbone using charged phosphoramidate analogues (29,30), by covalently attaching spermine derivatives to either cytosine N4 (31,32) or the 5[prime]-terminus (33) or 2[prime]-O (34,35) or by addition of an aminoethoxy group to the 2[prime] position (36,37). Each of these derivatives has been shown to produce a large increase in triplex stability. Interestingly, triplexes containing (N4-spermine)-5-methylcytosine are stable at pH 7.4 (31,32), even though N3 should be unprotonated at this pH, suggesting that the lack of the second hydrogen bond in the C+·GC triplet can be compensated by favourable electrostatic interactions. Other studies with syn-norspermidine linked to the 5-position of U in the third strand show significant triplex stabilisation at physiological pH (38).

To date most of the base analogues which have been prepared for triplex formation are derivatives of cytosine and have been designed to overcome the requirement for conditions of low pH. However, since C+·GC is more stable than T·AT, a different approach for increasing triplex stability is to modify the T·AT triplet so that its strength approaches that of C+·GC. In this paper we describe experiments with 5-propargylamino-dU (UP, Fig. 1a) as a charged analogue of thymine. This analogue bears a positive charge on the side group, rather than on the stacked ring system, and might form favourable ionic interactions with the phosphodiester backbone.


Figure 1. (a) Chemical structure of UP and thymidine (T). (b) Sequence of the short intramolecular triplex 5[prime]-A6-L-T6-L-UP5T (the linker, L, corresponds to two octanediol residues). (c) Sequences of intramolecular triplexes containing duplex overhangs and varying numbers of propargylamino (UP) residues. In each case the strands are linked by two octanediol residues. (d) Sequence of the 17 bp oligopurine tract in tyrT(43-59) (boxed) and its interaction with the three 9mer oligonucleotides (X = T or UP)

MATERIALS AND METHODS

Chemical synthesis

Phosphoramidite monomer for 5-(1-propargylamino)-2[prime]-deoxyuridine. 5[prime]-(4,4[prime]-dimethoxytrityl)-5-trifluoroacetylpropargyl-amino-2[prime]-deoxyuridine was synthesised as previously described (39, compound III). This was converted to the phosphoramidite by standard methods. To a solution of 5[prime]-(4,4[prime]-dimethoxy-trityl)-5-trifluoroacetylpropargylamino-2[prime]-deoxyuridine (300 mg, 0.44 mmol) in dry CH2Cl2 (3 ml) were added diisopropylethyl-amine (0.31 ml, 1.77 mmol) and 2-cyanoethyl N,N-diisopropyl chlorophosphine (0.12 ml, 0.49 mmol). This was stirred for 1 h at room temperature, followed by addition of CH2Cl2 (30 ml), washing with sat.KCl (30 ml), drying over Na2SO4 and removal of solvent in vacuo. Precipitation into cold hexane (-78°C) from CH2Cl2 and removal of solvent gave 5[prime]-(4,4[prime]-dimethoxytrityl)-5-trifluoroacetylpropargylamino-2[prime]-deoxyuridine-3[prime]-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite as an off-white foam (0.36 g, 93%). MS (ES+) 879.3.

DNA oligonucleotide synthesis

DNA oligonucleotides were synthesised on an Applied Bio-systems 394 solid phase DNA/RNA synthesiser on a 1.0 µmol scale. The unmodified phosphoramidite monomers, DNA solid supports and other reagents were purchased from Applied Biosystems. The synthesis was achieved using a standard cycle for the automated synthesiser. This ended with deprotection from the solid support with concentrated aqueous ammonia and base deprotection in concentrated ammonia heated at 55°C for 5 h. The oligonucleotides were then purified by reversed phase HPLC, desalted by NAP[trade]10 Sephadex G-25 columns, freeze dried and analysed by CZE. In order to confirm the integrity of propargylamino-dU containing oligonucleotide, the unpurified test sequence T7UPT7 was analysed by MALDI-TOF mass spectrometry. The observed molecular weight was 4536.50 (calculated 4537.50). The sequences of the oligonucleotides used in this study are presented in Figure 1b-d. For the intramolecular triplexes two octanediol residues (Oc) were used as a linker between each of the strands.

Chemicals and enzymes

Bovine DNase I was purchased from Sigma, and stored at -20°C at a concentration of 7200 U/ml. Restriction enzymes and reverse transcriptase were purchased from Promega. The naphthylquinoline triplex-binding ligand was a gift from Dr L. Strekowski, Department of Chemistry, Georgia State University, and was stored at -20°C as a 20 mM solution in dimethylsulphoxide.

UV melting

UV melting studies were performed as previously described (40) on a Perkin Elmer Lambda 2 UV/Vis spectrometer with PTP-1 temperature programmer or Perkin Elmer Lambda 15 UV/Vis spectrometer. Absorbances were monitored at 260 nm except for the propargylamino-dU containing oligonucleotides shown in Figure 1c which were measured at 285 nm. The heating rate was set to 1°C/min; a slower rate of 0.5°C/min did not alter the melting temperature (Tm), indicating the system was at equilibrium. Absorbance data were taken at intervals of 10 s, the absorbance melting curves were smoothed and the first derivative curves obtained using the Perkin Elmer PECSS2 software. Each UV melting experiment was repeated until three Tm values within 0.5°C were obtained. For temperatures <10°C, nitrogen was passed through the spectrophotometer to avoid condensation on the cuvette. Experiments at pH 5.0-6.5 were performed in 50 mM sodium acetate buffer containing 100 mM NaCl. Between pH 7.0 and 8.0 the buffer used was 50 mM sodium phosphate containing 100 mM NaCl, while at pH 8.5-9.0 the buffer was 50 mM borate containing 100 mM NaCl. The oligonucleotide concentration in the cell was ~3 µM, except for experiments performed at 285 nm for which the oligonucleotide concentration was ~9 µM.

DNA fragments

The tyrT DNA fragment was modified at positions 43 and 59 as previously described (28), generating the sequence shown in Figure 1. This was labelled by cutting with EcoRI and SmaI and labelled at the 3[prime]-end of the EcoRI site with [[alpha]-32P]dATP using AMV reverse transcriptase. The radiolabelled DNA fragment was separated from the remainder of the plasmid on a non-denaturing 6% (w/v) polyacrylamide gel. The isolated DNA fragment was dissolved in 10 mM Tris-HCl pH 7.5 containing 0.1 mM EDTA so as to give 10-20 c.p.s./µl as determined on a hand-held Geiger counter. For the quantitative footprinting experiments the absolute DNA concentration is not important, as long as it is much lower than the dissociation constant of the DNA binding ligand. We estimate that the strand concentration was <1 nM in all experiments.

DNase I footprinting

Radiolabelled DNA fragments (1.5 µl) were mixed with oligo-nucleotide (1.5 µl). Oligonucleotide concentrations refer to the concentrations in this mixture. Experiments at pH 5.0 and 6.0 were performed in 50 mM sodium acetate containing 10 mM MgCl2, while for pH 7.0 the buffer used was 10 mM Tris-HCl containing 50 mM NaCl and 10 mM MgCl2. The complexes were left to equilibrate for at least 3 h at 20°C. DNase I digestion was initiated by adding 2 µl DNase I dissolved in 20 mM NaCl containing 2 mM MnCl2 and 2 mM MgCl2. The reaction was stopped after 1 min by adding 4 µl of 80% formamide containing 10 mM EDTA and 0.1% (w/v) bromophenol blue.

Gel electrophoresis

The products of reaction were separated on 10% (w/v) polyacryl-amide gels containing 8 M urea (National Diagnostics). Gels, 40 cm long and 0.3 mm thick, were run at 1500 V for ~2 h. Gels were fixed in 10% (v/v) acetic acid before drying at 80°C and exposing to autoradiography at -70°C using an intensifying screen. Bands in the digestion pattern were assigned by comparison with Maxam-Gilbert sequencing lanes specific for adenine and guanine. For quantitative analysis gels were subjected to phosphor-imaging using a Molecular Dynamics STORM PhosphorImager.

Quantitative footprinting analysis

For quantitative analysis of the footprinting data the intensity of bands within the footprint was estimated using ImageQuant software. These were normalised for gel loading and digestion by comparing with a region for which DNase I cleavage was not affected by either the oligonucleotide or the triplex-binding ligand. Footprinting plots (41) were constructed from these data and C50 values, indicating the apparent oligonucleotide concentration which reduces band intensity in the region of the footprint by 50%, were derived by fitting a simple binding curve to plots of band intensity against oligonucleotide concentration using FigP for Windows (Biosoft). These were fitted to equation 1

Ic = I0 × C50/(L + C50) 1

where Ic is the band intensity in the presence of the ligand, I0 is the band intensity in the control, and L is the oligonucleotide concentration. The use of this equation to analyse footprinting data requires that the experiments are performed under conditions of single-hit kinetics and assumes that the DNA concentration is very low (much lower than the dissociation constant of the ligand).

RESULTS

UV-melting

UV-melting curves for the intramolecular triplex A6-L-T6-L-(UP)5T (Fig. 1b; L = two octanediol residues) were measured at different pHs and compared with the melting of the unmodified triplex A6-L-T6-L-T6. Under these buffer conditions (50 mM buffer plus 100 mM NaCl), both oligonucleotides showed a single sharp melting transition, suggesting that these triplexes are at least as stable as the underlying duplexes. The melting profile only separated into two transitions (triplex -> duplex, followed by duplex -> single strands) at much lower ionic strengths (not shown). At pH 7.0 the unmodified triplex melts at 314 K compared with 335.5 K for the triplex containing propargylamino-dU. Although the melting temperature of the unmodified triplex is pH-independent, as expected, the UP-containing oligonucleotide melts in a pH-dependent fashion (Fig. 2), displaying lower Tm values at higher pHs, so that at pH 9.0 the two oligonucleotides show very similar melting profiles. This pH dependency is consistent with the suggestion that the improved stability of the UP-containing structures is due to the positive charge on the amino group. Inclusion of propynyl-dU in place of UP in the Hoogsteen strand produces a melting profile with a transition temperature of 324.0 K (not shown), in between that of the T- and UP-containing oligonucleotides. This confirms that the increased stability of the UP-containing structures is not merely caused by stacking of the third strand propynyl residues, but is increased further by addition of the amino group.


Figure 2. Melting temperature of 5[prime]-A6-L-T6-L-(UP)5T (filled circles) (where the linker L is two octanediol residues) at different pHs. The experiments were all performed in 50 mM buffers containing 100 mM NaCl. The open circle shows the melting transition of A6-L-T6-L-T6 at pH 7.0.

We attempted to separate the two melting transitions (triplex -> duplex and duplex -> single strands) by using intramolecular triplexes containing a longer duplex portion (Fig. 1c), thereby increasing the temperature of the duplex -> single strands transition. At pH 7.0 the unmodified oligonucleotide P0 melts with two transitions at 295 and 335.5 K. This melting profile is not dependent on the pH. In contrast, the melting profile for oligonucleotide P2, containing two UP residues, depends on the pH conditions, as shown in Figure 3. The second transition (duplex -> single strand) shows little variation with pH, while the first transition (triplex -> duplex) shifts to higher temperatures at lower pHs (Fig. 3, inset). At pH 5.0 the triplex -> duplex transition occurs at 326.8 K compared with 314.7 K at pH 8.0. This pH dependency is further evidence that the improved stability of UP-containing triplexes is due to the positive charge on the amino group.


Figure 3. Melting profiles for oligonucleotide P2 at different pHs. The ordinate shows the first differential of the melting curve (dA/dT). Curve 1 corresponds to oligonucleotide P0, containing no modified bases, and was measured at 260 nm. Curves 2-5 correspond to oligonucleotide P2, containing two UP residues, and were measured at 285 nm. The measurements were made at pH 5.0 (curve 2), 6.0 (curve 3), 7.0 (curve 4) and 8.0 (curve 5). The inset shows the variation in Tm of P2 as a function of pH. The filled circles correspond to the triplex -> duplex transition, while the open circles correspond to duplex -> single strands.

We also examined how the number of UP substitutions affects the melting profile at pH 7.0, using the series of intramolecular triplexes shown in Figure 1c. The results of these UV-melting experiments are summarised in Table 1. As expected the duplex -> single strand transition (Tm2) is not affected by the number of substitutions, whereas Tm1 increases by ~12-15 K with each additional substitution. With three and six UP substitutions (oligonucleotide P3 and P6) the complex melts with a single transition.


Table 1. Tm for oligonucleotides P0, P1, P2, P3 and P6 (containing different numbers of propargylamino-dU residues) measured at pH 7.0
Tm1 corresponds to the triplex -> duplex transition, while Tm2 corresponds to duplex -> single strand. Where only one value is shown the two transitions could not be resolved and the data were described by a single melting transition.


Footprinting

We have previously targeted the 17mer oligopurine tract in tyrT(43-59) with three different T-containing 9mer oligonucleotides (Fig. 1d) and have shown that third strand affinity increases with increasing numbers of C+·GC triplets (28). Indeed the upper oligonucleotide, generating a complex with eight T·AT triplets and one C+·GC, failed to produce a DNase I footprint at concentrations as high as 30 µM. These results were taken as evidence that isolated C+·GC triplets impart a greater triplex stability than T·AT. We have therefore repeated these experiments with UP-containing oligonucleotides. The results for experiments performed at pH 5.0 are presented in Figure 4. Looking first at the third strand containing a single cytosine and seven UP residues (upper oligonucleotide in Fig. 1d), which is targeted at the 3[prime]-(lower) end of the oligopurine tract, a clear footprint can be seen which covers the intended site and which persists to an oligonucleotide concentration of [le]5 nM (Fig. 4, left hand panel). As commonly observed, this footprint is accompanied by enhanced cleavage at the 3[prime]-end of the target site at the triplex-duplex junction, an observation which is taken as evidence for a local oligonucleotide-induced DNA structural change. The intensity of this enhancement also varies with the oligonucleotide concentration and persists to at least 2 nM. Quantitative analysis of these data yielded C50 values of 1.5 nM for the protection and 0.6 nM for the enhanced cleavage. These results, together with the results for the other oligonucleotides used in this study, are summarised in Table 2. Since the unmodified T-containing oligonucleotide failed to produce a DNase I footprint, even at concentrations as high as 30 µM, it is clear that replacement of the seven thymidines with propargyl-amino-dU has increased the affinity by at least four orders of magnitude.


Table 2. Summary of C50 values (nM) for the interaction of 9mer T- or UP-containing oligonucleotides with the 17mer oligopurine target site in tyrT(43-59)
Data for T-containing oligonucleotides are taken from (28). NO, no footprint detected with 30 µM oligo-nucleotide. n.d., not determined. *values determined from enhanced DNase I cleavage at the triplex-duplex junction.


Figure 4. DNase I cleavage patterns of tyrT(43-59) in the presence of varying concentrations of the three propargylamino-dU-containing oligonucleotides at pH 5.0. For the first two panels, the oligonucleotide concentrations decrease from left to right with concentrations of 1, 0.5, 0.2, 0.1, 0.05, 0.01, 0.005, 0.002, 0.001, 0.0005, 0.0002 and 0.0001 µM. For the third panel the oligonucleotide concentrations were 6.7, 3.3, 1.3, 0.67, 0.33, 0.13, 0.067, 0.033, 0.013, 0.0067, 0.0033, 0.0013, 0.000, 0.0007, 0.0001 and 0.00007 µM. The numbers alongside the first panel correspond to the sequence numbering scheme used in previous publications. Tracks labelled ‘con’ show digestion of the DNA in the absence of added oligonucleotide. Tracks labelled ‘GA’ are Maxam-Gilbert markers specific for purines. The brackets indicate the positions of the target sites for each of the 9mer oligonucleotides. The reactions were performed in 50 mM sodium acetate pH 5.0 containing 10 mM MgCl2.


The middle panel of Figure 4 shows DNase I footprints for the interaction with the oligonucleotide containing two cytosines and six UP residues (middle oligonucleotide in Fig. 1d), which is targeted at the centre of the oligopurine tract. This oligonucleotide produces a clear footprint at its target site which persists to a concentration of <0.1 µM, and which is also accompanied by a weak region of enhancement at the 3[prime]-(lower) end. Quantitative analysis of these data yield a value of 12 nM for C50, which compares with 4.7 µM for the unmodified T-containing oligonucleotide. Once again, replacement of T by UP has caused a large increase in triplex stability. The right hand panel shows similar data for the oligonucleotide containing four cytosines and four UP residues (lower oligonucleotide in Fig. 1d), which is targeted at the 5[prime]-(upper) end of the oligopurine tract. This produces a footprint at its intended target site which persists to a concentration of <0.1 µM. Quantitative analysis of these data yield a C50 value of 3.8 nM, which compares to 130 nM for the unmodified oligonucleotide. In this instance the substitution has had a smaller though significant effect on triplex stability. These C50 values are summarised in Table 2.

Figure 5 shows the results of similar experiments with the oligonucleotides containing one and two cytosines performed at pH 6.0 and 7.0. At pH 6.0 the oligonucleotides containing one and two cytosines generate footprints at concentrations of ~0.5 and 10 µM, respectively. The footprint with the oligonucleotide containing one cytosine is accompanied by enhanced cleavage at the 3[prime]-(lower) end of the target site, as seen at pH 5.0. Quantitative analysis of these data yields C50 values of 56 and 70 nM for oligonucleotide 5[prime]-(UP)6CUPT, estimated from the footprint and enhancement respectively and 8.8 µM for oligonucleotide 5[prime]-UPUPCUPCUPUPUPT. At pH 7.0 (right hand panel) the footprints are much weaker. The oligonucleotide containing two cytosines shows almost no changes in the cleavage pattern, while the one generating only one C+·GC triplet shows attenuated cleavage at the highest concentrations. Quantitative analysis of the data with 5[prime]-(UP)6CUPT yields a C50 value of 1.8 µM. Although these complexes are less stable than those formed at pH 5.0 they are still a great improvement on the unmodified T-containing oligonucleotides for which no footprints were observed at [ge]pH 6.0. Since these oligonucleotides generate complexes which contain one and two C+·GC triplets, their lower stability at elevated pHs reflects a combination of the lack of protonation of C as well as any effect on the charge on the terminal amino moieties.


Figure 5. DNase I cleavage patterns of tyrT(43-59) in the presence of varying concentrations of the propargylamino-dU-containing oligonucleotides at pH 6.0 and 7.0. For each experiment the oligonucleotide concentrations decrease from left to right with concentrations of 30, 20, 10, 5, 2, 1, 0.5, 0.2 and 0.1 µM at pH 6.0 and 30, 10, 5, 2, 1, 0.5, 0.2 and 0.1 µM at pH 7.0. Tracks labelled ‘con’ show digestion of the DNA in the absence of added oligonucleotide. Tracks labelled ‘GA’ are Maxam-Gilbert markers specific for purines. The brackets indicate the positions of the target sites for each of the 9mer oligonucleotides. The reactions were performed in 50 mM sodium acetate pH 6.0 containing 10 mM MgCl2.

We have also studied the effect of modifying only three or four of the seven Ts in the oligonucleotide generating one C+·GC triplet. The results are presented in Figure 6. It can be seen that the oligonucleotide containing only three UP residues does not produce a clear footprint, though some enhanced cleavage is evident at the lower end of the target site. The oligonucleotide containing four UP residues causes attenuated DNase I cleavage at the highest concentrations (50 and 30 µM), which is also accompanied by enhanced cleavage. Quantitative analysis of the results with 5[prime]-UPTUPTUPTCUPT yields C50 values of 21 and 4.3 µM from the footprint and enhancement, respectively. It should be noted that, although these oligonucleotides generate triplexes with much lower stability than 5[prime]-(UP)6CUPT, the complexes are more stable than those formed with the T-containing oligonucleotide which failed to produce a footprint under any conditions. The dramatically weaker binding of these oligonucleotides was surprising. We therefore checked their potential for forming stable triplexes by adding 10 µM of the naphthylquinoline triplex-binding ligand (42), which has been show to potentiate triplex formation with the T-containing oligonucleotides (28). The results (not shown) reveal clear footprints with both oligonucleotides which persist to concentrations <10 nM.


Figure 6. DNase I cleavage patterns of tyrT(43-59) in the presence of varying concentrations of 5[prime]-UPTTUPTTCUPT and 5[prime]-UPTUPTUPTCUPT. For each oligonucleotide the concentrations decrease from left to right with concentrations of 50, 30, 20, 10, 8, 5, 2 and 1 µM. Tracks labelled ‘con’ show digestion of the DNA in the absence of added oligonucleotide. Tracks labelled ‘GA’ are Maxam-Gilbert markers specific for purines. The brackets indicate the positions of the target sites for each of the 9mer oligonucleotides. The reactions were performed in 50 mM sodium acetate pH 5.0 containing 10 mM MgCl2.

DISCUSSION

The results presented in this paper demonstrate that oligonucleotides containing propargylamino-dU generate much more stable triplexes than those containing thymine and that this difference is greatest at lower pHs. At pH 7.0, A6-L-T6-L-(UP)5T melts ~20 K higher than A6-L-T6-L-T6, compared with a 24 K difference at pH 5.0. Quantitative footprinting experiments show that at pH 5.0 (UP)6CUPT binds to its target site at least four orders of magnitude better than T6CTT. This oligonucleotide binds much less well at pH 7.0, though this difference includes a contribution from the pH dependence of the C+·GC triplet. Nonetheless, at pH 7.0 it still binds better than the unmodified oligonucleotide, which fails to produce a DNase I footprint. We presume that the increased stability of UP-containing triplexes arises from favourable ionic interactions between the charged amino group and the negatively charged phosphodiester backbone. Previous studies have shown that propynyl-dU also increases triplex stability as a result of the stacking interactions between adjacent propynyl residues (43,44). Although this stacking effect may be important for the increased stability of UP-containing triplets, it cannot be the only factor since we find that, at low pHs, propynyl-dU containing oligonucleotides melt ~10 K lower than those containing propargylamino-dU. In addition an effect from stacking of the propynyl groups does not account for the observed pH dependency of triplex formation in both the UV-melting and footprinting experiments.

Substitution of thymidine with propargylamino-dU does not appear to have affected the stringency of triplex formation; in each case triplexes are only observed at the intended target sites. Although addition of the positively charged arms has increased triplex stability, the specificity of the interaction still arises from the formation of the correct Hoogsteen base pairs. Within the range of concentrations employed in the footprinting experiments, the additional positive charge has not promoted the formation of any unusual triplets such as UP.GC.

The stabilisation of triplexes by including UP is clearly different from the greater stability imparted by C+·GC compared with T·AT. Although triplex stability increases with the number of isolated C+·GC triplets (15,27,28), adjacent C+·GC triplets cause a decrease in stability (45), presumably due to charge repulsion between the positively charged cytosines within the stacked bases. In contrast, the stability of UP·AT containing triplexes is not decreased by the presence of adjacent charged residues. 9mer oligonucleotides containing three or four UP substitutions formed triplexes which, although more stable than the unsubstituted oligonucleotide, were much less stable than the triplex containing seven UP-residues. We presume that the ability to form adjacent UP·AT triplets is because the positive charge on each UP-residue is screened by the negatively charged phosphodiester backbone. In addition, adjacent UP·AT triplets will benefit from stacking interaction between the propynyl groups.

The increased stability of parallel triplexes containing charged analogues of thymine opens the possibility of adding similar groups to other bases so as to further increase the stability of C+·GC, or improve the stability of antiparallel triplexes. The use of UP as a triplex base analogue may be limited as a result of its pH dependency, though it may prove useful in combination with cytosine analogues which recognise GC pairs in a pH dependent fashion. Further analogues containing permanent positive charges will overcome this limitation.

ACKNOWLEDGEMENT

This work was supported by grants from the Cancer Research Campaign.

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*To whom correspondence should be addressed. Tel: +44 1703 594374; Fax: +44 1703 594459; Email: krf1@soton.ac.uk

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