Recognition of Hairpin-Containing Single-Stranded DNA by

Bioconjugate Chem. 1999, 10, 439−446

439

Recognition of Hairpin-Containing Single-Stranded DNA by Oligonucleotides Containing Internal Acridine Derivatives Jean-Christophe Franç ois* and Claude He´le`ne Laboratoire de Biophysique, Museúm National d'Histoire Naturelle, INSERM Unite´ 201- CNRS UA 481, 43 rue Cuvier, 75231 Paris Cedex 05, France . Received October 7, 1998; Revised Manuscript Received January 27, 1999

Oligodeoxynucleotides with an internal intercalating agent have been targeted to single-stranded sequences containing hairpin structures. The oligonucleotide binds to nonadjacent single-stranded sequences on both sides of the hairpin structure in such a way as to form a three-way junction. The acridine derivative is inserted at a position that allows it to interact with the three-way junction. The melting temperature (Tm) of complexes formed between the hairpin-containing target and oligonucleotides containing one internal acridine derivative was higher than that obtained with the same target and an unmodified oligonucleotide (∆Tm ) +13 °C). The internal acridine provided the oligonucleotide with a higher affinity than covalent attachment to the 5′ end. Oligonucleotides could also be designed to recognize a hairpin-containing single-stranded nucleic acid by formation of Watson-Crick hydrogen bonds with a single-stranded part and Hoogsteen hydrogen bonds with the stem of the hairpin. An internal acridine derivative was introduced at the junction between the two domains, the double helix domain with Watson-Crick base pairs and the triple helix domain involving Hoogsteen base triplets in the major groove of the hairpin stem. Oligonucleotides with an internal acridine or an acridine at their 5′ end have similar binding affinities for the stem-loop-containing target. The bis-modified oligonucleotide containing two acridines, one at the 5′ end and one at an internal site, did not exhibit a higher affinity than the oligonucleotides with only one intercalating agent. The design of oligonucleotides with an internal intercalating agent might be of interest to control gene expression through recognition of secondary structures in single-stranded targets.

INTRODUCTION

Hairpin structures frequently occur in RNA secondary structures and also in DNA sequences that are found near functional loci and therefore seem to play a critical role in gene regulation (1). In the antisense strategy where the antisense oligonucleotides are targeted to single-stranded messenger RNAs, it was shown that stem-loop structures can compete with intermolecular hybridization of the oligonucleotides to the complementary sequences (2). Nevertheless, hairpin structures could also be exploited in the design of the recognition elements of an oligonucleotide aimed at inhibiting mRNA translation (3, 4) or probing RNA structures (5). We have previously shown that hairpin-containing nucleic acids could bind oligodeoxynucleotides complementary to nonadjacent single-stranded sequences on both sides of the stem-loop sequence (6). Oligonucleotides can also bind to hairpin-containing single-stranded nucleic acids (DNA or RNA) by using both triple helix and double helix formation (3, 7-15). In the present paper, we have investigated the effect of introducing an intercalating agent, an acridine derivative, in an oligonucleotide designed to recognize two single-stranded sequences separated by a hairpin structure. We demonstrate that when the acridine is inserted at an internal position close to the bases of the stem loop structure, the resulting complexes are more stable than when it is linked to the 5′ end of the oligonucleotide, * To whom correspondence should be addressed. Phone: 331-40-79-37-08. Fax: 33-1-40-79-37-05. E-mail: francois@cimrs1. mnhn.fr.

suggesting a preferential site of interaction for the acridine ring near the center of the three-way structure. The formation of triple helices is of interest in both the antisense and the antigene strategies (16). Clamp oligonucleotides can bind to an oligopurine sequence in a single-stranded nucleic acid by forming both WatsonCrick and Hoogsteen hydrogen bonds (17-19). Circular oligonucleotides can also be designed to bind via both double and triple helix formation (20, 21). Recently, it was shown that the incorporation of an acridine derivative at an internal site of an oligonucleotide stabilizes triplexes with an interruption in the oligopurine target sequence and therefore opens new possibilities to extend the range of DNA sequences recognized by triplexforming oligonucleotides (22, 23). Triplex-specific intercalating agents such as benzopyridoindole and benzopyridoquinoxaline derivative could also be introduced into triplex-forming oligonucleotides to increase binding affinity to the DNA double helices (24, 25). We have previously demonstrated that antisense oligonucleotides could bind to hairpin-containing singlestranded nucleic acids by using both triple helix and double helix formation (9). These oligonucleotides interact with one of the two single-stranded sequences flanking the hairpin via Watson-Crick base pair formation and with the double-stranded stem sequence of the hairpin via Hoogsteen base triplet formation. These newly designed antisense oligonucleotides were called WatsonCrick/Hoogsteen oligonucleotides (abbreviated as WC/H). We have now synthesized WC/H oligonucleotides with an internally incorporated acridine in such a way that the acridine ring is located at the junction between the

10.1021/bc9801225 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/26/1999

Franç ois and He´le`ne

440 Bioconjugate Chem., Vol. 10, No. 3, 1999

Figure 1. Chemical structures of the internally incorporated acridine within an oligonucleotide and the acridine linked at the 5′ end of an oligonucleotide. Note that the acridine ring covalently linked to the 5′ end was noted in the text as Acr and that the internal acridine derivative was referred to as X. B represents a base.

duplex and triplex portions of the complex. These WC/H oligonucleotides with an internal acridine form more stable complexes with their target than the unmodified oligonucleotides. The binding affinity of the WC/H oligonucleotide with an internal acridine is similar to the binding affinity of the oligonucleotide with an acridine linked to its 5′ end.

plastic sheets silica gel 60 F254, Merck), and the fluorescence of the incorporated acridine (obtained after excitation at 320 nm). Dark bands at 254 nm (corresponding to absorption of the bases of the oligomers) comigrated with yellow bands at 320 nm (corresponding to the fluorescence of the acridine). With this control assay, all acridine-containing oligonucleotides exhibited only one band that migrated more slowly than the dark band of the unmodified oligonucleotides. The length of 5′ end-labeled unmodified oligonucleotides has also been checked by gel electrophoresis. The concentration of oligonucleotides and acridinecontaining oligonucleotides was determined using the extinction coefficient for acridine at 424 nm, 8500 M-1 cm-1 or calculated by nearest-neighbor method (26) assuming an extinction coefficient for acridine of 85 000 M-1 cm-1 at 260 nm. The concentration of the oligonucleotide containing two acridine rings was estimated at 260 nm. With this oligonucleotide, calculations using the extinction coefficient at 424 nm gave an underestimated concentration, probably due to the existence of interactions involving the two acridine rings. Melting Experiments. Absorption spectra were recorded on an UVIKON 940 spectrophotometer with cell holders thermoregulated by a circulating liquid (80% water, 20% ethylene glycol). Sample temperature was decreased from 90 to 5 °C and increased back to 90 °C at a rate of 0.25 °C/min with absorption readings at 260 and 540 nm taken every 5 min. Melting curves were obtained by subtracting the absorbance recorded at 540 nm where none of the constituents absorbed light from that recorded at 260 nm. Under all the experimental conditions described in the figures, all melting profiles were reversible. The melting temperature Tm ((1 °C) was determined as the maximum of the first derivative of melting curves. All the Tm experiments presented in this study were performed with 2 µM hairpin-containing target and 2 µM oligonucleotide ligand, therefore, enabling direct comparison of Tm values. It should be noted that these Tm values depend on the concentration of oligonucleotides. For acridine-containing oligonucleotides, identical melting temperatures were obtained from absorbance measurements at 260 or 425 nm (where acridine absorbs).

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Oligonucleotides. Oligonucleotides and acridinecontaining oligonucleotides were synthesized by Eurogentec (Belgium). Acridine phosphoramidite (Glen Research) was either internally incorporated into oligonucleotides in the course of the synthesis or incorporated at the end of synthesis for 5′-linked oligonucleotides. Note that the natural three carbon internucleotidic phosphate distance is maintained in the oligonucleotide containing an internal acridine (Figure 1). The acridine ring was tethered to the backbone via five atoms. The 5′-linked acridine was tethered to the 5′-phosphate group via a seven atom containing linker. A bis-modified oligonucleotide with two acridines, one internal and one linked to the 5′ end, was obtained by the same procedure. Before utilization, the acridine-oligonucleotide conjugates were purified by gel-filtration on G25 sephadex minicolumns (Boehringer Mannheim) in order to eliminate uncoupled acridine. The quality of acridine-oligonucleotide conjugates was then checked by denaturing gel electrophoresis (20% polyacrylamide/7 M urea). For detection, we used both the UV shadow effect of the oligonucleotide with irradiation at 254 nm on a chromatography paper containing a fluorescent-indicator, (TLC

Design of Hairpin-Containing Targets and Acridine-Containing Oligonucleotides. In the first DNA model, a single-stranded nucleic acid fragment, 36 nucleotides (nt) in length (36mer) was used as a target for 16mer oligonucleotides linked to an acridine derivative (Figure 2A). The structure of the acridine conjugates is shown in Figure 1. The 36mer can form a hairpin structure with four G‚C and two A‚T base pairs in the stem and four thymines in the loop. The sequence of the control 35mer was identical to that of the 36mer except that the sequence in the stem-loop 5′CGATCGTTTTCGATCG3′ was changed to 5′CTCCAATTCTTC3′, preventing the formation of a hairpin structure in the 35mer. Three additional nucleotides (AGT) were present at the 3′ end of the 35mer [referred in a previous paper as 35mer-(AAT) (6)]. The 16mer oligonucleotides were designed to bind to both sides of the hairpin-containing 36mer forming two mini double helices separated by the stem-loop region (Figure 2A). For comparison with targets containing intervening sequences, oligonucleotides and acridine-oligonucleotide conjugates were targeted to the 23mer, a single-stranded DNA target with no hairpin structure (Figure 2B).

Recognition of DNA-Hairpin by Acridine-Oligonucleotides

Figure 2. (A) Sequences of the hairpin-containing oligonucleotide 36mer and of the 35mer with no hairpin structure. The unmodified ligands, 16mer, 17mer, 18mer, and 19mer together with the sequences of the acridine-modified oligonucleotides are shown. X corresponds to the internal acridine and Acr to the 5′-linked acridine (see Figure 1). (B) Sequences of the 23mer, 24mer, 25mer, and 26mer targets that exhibited no hairpin structure. Sequences of their unmodified or modified oligonucleotide ligands, 16mer, 16mer-X, and Acr-16mer are shown in Figure 2A.

In the second DNA model, the target 46merWCfH has two different domains, a double-stranded stem and a single-stranded sequence, allowing both triple helix and double helix formation with acridine-containing 17mer oligonucleotide ligands (Figure 3). Only one possibility for designing the WC/H targets was studied [see previous paper for details (9)]. The single-stranded purine-rich region of the 46mer was chosen to be on the 5′ side of the hairpin and was referred to as the Watson-Cricktoward-Hoogsteen orientation, 46merWCfH in ref 9. The 17mer oligonucleotides were designed to bind to both single-stranded and double-stranded regions of the hairpin-containing 46merWCfH. The stem sequence in 46merWCfH was chosen to be long enough to give rise to two distinct melting transitions corresponding to oligonucleotide dissociation and stem disruption. It should be noted that the 46merWCfH had a short (5 nt) singlestranded sequence added to the 3′ end of the stem. Acridine derivatives were either attached to the 5′ end of the oligonucleotides (Acr-16mer and Acr-17mer) or incorporated in the center of the oligonucleotide sequence (for 16mer-X, 17mer-X, and 18mer-X). The acridine of the oligonucleotide ligand was located in the complex formed with the DNA targets, either between the two singlestranded parts of the 36mer (Figure 2) or at the junction between the single-stranded and the double-stranded domains of 46merWCfH (Figure 3). Additional thymines were introduced in replacement of the acridine derivative

Bioconjugate Chem., Vol. 10, No. 3, 1999 441

Figure 3. Sequences of the hairpin-containing oligonucleotide 46merWCfH and its ligands: unmodified 17mer, 18mer, 19mer, and 16mer; acridine-containing ligands: 17mer-X, 18mer-TX, 18mer-XT, 16mer-X, and Acr-17mer. Note that single-stranded sequences are present on both the 5′ and 3′ sides of the hairpin of the 46mer WCfH. The mutated oligonucleotide 17mer-Mut 5′dTTCACTCTTCTCTTTCC3′ is identical to the 17mer sequence except for three nucleotides located in the 5′ part of the oligonucleotide (underlined on the ligand sequence). The 16mer and 16mer-X 5′dTTTTTTCTCTCTTTCC3′ lack an internal thymine when compared to the 17mer and 17mer-X, respectively.

or near the acridine derivative to obtain 17mers, 18mers, 19mers, 18mer-TX, and 18mer XT (Figures 2 and 3). A bis-modified oligonucleotide, Acr-17mer-X, with two acridines (one at an internal position and one at the 5′ end of the oligonucleotide) was also targeted to the 36mer (Figure 2A). Recognition of Two Nonadjacent Sequences by Acridine-Oligonucleotide Conjugates: Model 1. As shown on the melting profile presented in Figure 4A, the melting transitions corresponding to oligonucleotide dissociation and stem fusion of the 36mer were well separated. Melting temperature (Tm) of the stem-loop structure of the 36mer was estimated from the first derivative of melting curves as shown in Figure 4B. The Tm of the 5′ CGATCGTTTTCGATCG3′ stem loop in the 36mer was 65 ( 1 °C in the incubation conditions of Figure 4A (0.1 M NaCl). Previous melting experiments have shown that the 5′CGATCGTTTTCGATCG3′ oligonucleotide formed a hairpin structure with a Tm of 70 °C in 0.1 M NaCl (27). These results suggested that the addition of flanking sequences to the hairpin-forming sequence 5′CGATCGTTTTCGATCG3′ decreased by 5 °C the stability of the hairpin in the 36mer. The unmodified 16mer oligonucleotide binds to the 36mer with a Tm of 25 ( 1 °C at 2 µM concentration in a pH 7.02 buffer containing 50 mM cacodylate and 0.1 M sodium chloride (Figure 4). The addition of an acridine at the 5′ end of the 16mer in Acr-16mer increased the Tm value by 8 °C (Figure 5). This stabilization was already demonstrated for oligonucleotide-acridine conjugates targeted to single-stranded DNA sequences without any secondary structure (28, 29). The 16mer-X with an internal acridine in the center of the 16mer sequence had a higher Tm value (38 ( 1 °C) as compared to the Acr-16mer (Tm ) 33 °C) and 16mer (Tm ) 25 °C) (Figure

442 Bioconjugate Chem., Vol. 10, No. 3, 1999


Figure 5. Schematic representations of the complexes formed by acridine-containing and unmodified oligonucleotide ligands (L) with the hairpin-containing 36mer or the 35mer target containing no hairpin structure (T), 36mer and 35mer. Melting temperatures indicated below each complex were obtained with 2 µM/2 µM T/L in the same buffer used in Figure 4. The Tm of the 16mer/36mer (Tm ) 25 °C) was taken as reference for the ∆Tm calculations.

Figure 4. (A) Melting curves measured at 260 nm for 2 µM 36mer bound to 2 µM of 16mer (9), 16mer-X (b), Acr-16mer (4), and 17mer (O) in 50 mM cacodylate buffer, pH 7.02, containing 0.1 M NaCl. The melting temperature corresponding to stem fusion is 65 ( 1 °C. (B) First derivative of the melting curves shown in Figure 4A. Legend symbols are the same as in Figure 4A. In addition the derivative of the melting curve of the 36mer/ 18mer complex is also shown ([).

5). These results suggested that the location of the acridine in the 16mer-X/36mer complex, near the branchpoint of the three-way structure, was likely a preferential site for intercalation of acridine. The replacement of acridine by one internal thymine (17mer/36mer complex) at the junction between the two single-stranded domains of the 36mer strongly decreased the Tm value from 38 to 28 °C. The 17mer/36mer complex was only slightly stabilized (∆Tm ) +3 °C) as compared to that for the 16mer/36mer complex (Figure 5). The 18mer with two additional thymines had a Tm increased by 6 °C (Tm ) 31 °C) (Figure 5), indicating that the introduction of thymines in such structures improved the binding affinity likely via stacking interactions at the branch point as previously described for DNA three-arm junctions (30-33). No additional increase of stability was

observed with the introduction of a third thymine (Tm ) 30 °C for the 19mer). The 17mer-X oligonucleotide with an acridine and a thymine both located at the junction of the three-way structure has a higher melting temperature than the unmodified 17mer [∆Tm (17mer-X/17mer) ) +9 °C], but has a melting temperature similar to that of Acr-17mer. The Tm values for complexes with 17mer-X and Acr-17mer are, respectively, 37 and 36.5 °C (data not shown). This result indicated that the addition of acridine either internally or at the 5′ end of the oligonucleotide ligand stabilized to the same extent as the complexes containing one unbound internal thymine (17mer). It should be noted that the replacement of the central thymine in the 17mer by an acridine ring (to obtain the 16mer-X) strongly increased binding affinities as shown by the ∆Tm between the complexes formed by 17mer and 16mer-X [∆Tm (16mer-X/17mer) ) +10 °C] (Figure 5). This result indicated that the tricyclic acridine ring interacted as an intercalating agent much better than an additional thymine. The charge of the acridine molecule (assuming that at neutral pH, the ring is still positively charged) may also participate in stabilization of complexes. Nevertheless, this electrostatic stabilization may occur with internal acridine and with acridine linked to the 5′-end of the oligonucleotide ligand. The addition of one thymine in 17mer-X/36mer or two thymines in 18mer-TX/ 36mer did not give more stable complexes than that formed by 16mer-X/36mer [Tm (17mer-X/36mer) ) 37 °C and Tm (18mer-TX/36mer) ) 38 °C]. In an attempt to increase the binding affinity of 17mer-X ligand to its 36mer target, we have synthesized Acr-17mer-X, an oligonucleotide with two acridine derivatives, one inserted at a internal site and the second one at the 5′ end (Figure 2). Surprisingly, no additional stabilization was observed when compared to the oligo-



Figure 6. Schematic representations of the complexes formed by acridine-containing and unmodified oligonucleotide ligands (L) with the targets that contains no secondary structures (T), 23mer, 24mer, 25mer and 26mer. Experimental conditions were the same as in Figures 4 and 5. The two columns (to the right of the figure) represent the ∆Tm values with and without acridine attached in the center or at the 5′-end of the ligand for each of the target.

nucleotides with only one acridine, 17mer-X and Acr17mer [Tm (Acr-17mer-X/36mer) ) 36 °C]. The Acr-17mer-X did not give a melting transition when heated alone at 5 µM in the conditions described in Figure 4A (data not shown). However, this does not exclude a stacking interaction between the two acridine rings. 2-Methoxy-6-chloro-9amino acridine derivatives are known to self-associate in aqueous solutions with a dimerization constant around 103 M-1 (34). In the oligonucleotide, this process becomes intramolecular and can compete with binding of the bissubstituted oligonucleotide to its target sequence. To determine if the presence of the hairpin structure was necessary for the increase in binding stability of oligonucleotides with an internal acridine, the 35mer which has no hairpin structure but contains the target sequences for each half of the 16mers was synthesized. As shown in Figure 5, the absence of hairpin structure in the 16mer/35mer complexes slightly decreased Tm values, but the addition of acridine either at the 5′ end or internally increased melting temperatures to the same extent [∆Tm(16mer-X/16mer) ) +8.5 °C and ∆Tm(Acr-16mer/16mer) ) +8 °C]. As compared to the hairpin-containing structure [∆Tm(16mer-X/16mer) ) +13 °C and ∆Tm(Acr16mer/16mer) ) +8 °C], there is clearly an increased binding energy of the internal acridine with the hairpin-containing target reflecting the strongest interaction of the acridine with the three-way junction. Double Helices Formed by Acridine-Oligonucleotide Conjugates and Target Oligonucleotides without Any Secondary Structure. Oligonucleotides and acridine-oligonucleotide conjugates were targeted to the 23mer, a single-stranded DNA target with no secondary structure and no interruption of the complementary sequence of the 16mer and to longer oligomers, 24-26 nt in length, with one, two, or three additional bases at the position where the hairpin sequence of the 36mer was inserted (Figure 2B). All melting temperatures described in Figure 6 were higher than those obtained with targets containing hairpin structures (Figure 5), emphazing the destabilizing role of the hairpin structure in complexes with 36mer. As shown in Figure 6, when the 16mer was targeted successively to the 23mer, 24mer, 25mer, and 26mer, a decrease of Tm values was observed, demonstrating the destabilizing effect of introducing bulges, i.e.,

nonmatched additional bases (35, 36). Incorporation of an internal acridine in the 16mer sequence (16mer-X) resulted in an increase in binding affinity but to a lesser extent (∆Tm(16mer-X/23mer) ) +4 °C, see Figure 6) than that obtained with the 36mer (∆Tm(16merX/36mer) ) +13 °C, see Figure 5). Similar results were obtained with oligonucleotides containing an internal acridine ring that were targeted to a target containing an abasic, an apyrimidinic or an apurinic site (37, 38). In contrast to what was observed with the 16mer/ 36mer complexes (Figure 5), the Acr-16mer oligonucleotide had a higher Tm value than the 16mer-X when bound to the 23mer (∆Tm ) +7 °C). It should be noted that the stabilization obtained when the acridine was attached to the 5′ end of the 16mer (Acr-16mer) was the same for all targets: 23mer, 24mer, 25mer, and 26mer (Figure 6). For all targets in Figure 6, ∆Tm(Acr-16mer/16mer) was +7 °C. For the 36mer and the 35mer, addition of acridine at the 5′ end resulted in an increase of Tm of +8 °C (Figure 5). These results demonstrated that the intercalating acridine exhibited a similar behavior concerning the stabilization of duplex DNA even if this DNA contained bulged nucleotides or hairpins located 8 nucleotides away from the 5′ end of the oligonucleotide ligand. The addition of an acridine ring in the center of the 16mer sequence (16mer-X) increased Tm values by +4, +11, and +9 °C for the 16mer-X/23mer, 16mer-X/24 or 25mer, and 16mer-X/26mer, respectively (Figure 6). A stabilization of +8.5 °C was obtained with 16mer-X bound to the 35mer target [(∆Tm(16mer-X/16mer) ) +8.5 °C, see Figure 5], suggesting that the additional stabilization afforded by an internally attached acridine does not depend on the number of looped-out bases above three bases. The stabilization observed when the 16mer-X binds to the hairpin-containing 36mer (∆Tm) +13 °C) indicates that the hairpin is formed when the oligonucleotide is bound and that the acridine has a preferential site of interaction at the branch point of the three-way junction. Recognition of Hairpin-Containing Structures by Acridine-Oligonucleotide Conjugates via Both Watson-Crick and Hoogsteen Hydrogen Bonding: Model 2. We previously demonstrated that the 17mer


444 Bioconjugate Chem., Vol. 10, No. 3, 1999 Table 1: Melting Temperatures (Tm) for the Dissociation of Oligonucleotide Ligands (L) from a Hairpin-Containing Target (T), 46Mer WCfHa oligonucleotide ligands

Tm (S+) (°C)

∆Tm (S+)

Tm (°C)

∆Tm

17mer 17mer-X Acr-17mer Acr-17mer-X 18mer 18mer-TX 19mer 17mer-Mut

34 50 49 46 42 50 39 26.5

0 +16 +15 +12 +8 +16 +5 -7.5

14 35 31.5 31.5 24 34 19 12.5

0 +21 +17.5 +17.5 +10 +20 +5 - 1.5

a See sequences in Figure 3. Concentration of both the oligonucleotide ligand and the target were 2 µM. Samples were incubated in a 50 mM cacodylate buffer containing 0.1 M NaCl at pH 7.02 in the absence (right columns) or in the presence of 0.2 mM spermine (S+) (left columns). Tm values were determined as described in Materials and Methods and were rounded off to the nearest 0.5 °C and error is estimated at ( 1 °C. Melting temperatures of the stem sequence of the 46mer WCfH are respectively 64 °C and 70 °C in absence and in the presence of 0.2 mM spermine. ∆Tm represents the increase in Tm upon addition of thymines or acridine derivatives for each incubation conditions. The reference is the 17mer complex.

shown in Figure 3 binds to the 46mer WCfH by forming both Watson-Crick and Hoogsteen hydrogen bond interactions (9). The difference in stability between the 17mer and the 17mer-Mut complex (∆Tm ) -7.5 °C, Table 1) demonstrated that the 5′ end of the 17mer was bound to the stem sequence of the 46merWCfH via triple helix formation as previously described (9). In the 18mer/46merWCfH complex, the addition of a thymine at the junction of the two domains increased the Tm by +8 °C (Figure 7A and Table 1). As previously reported (9), the addition of one nucleotide increases the binding affinity when it is located at the junction of a WCfH complex, but decreased the affinity when it was located at the junction of a HfWC complex (9). The addition of two thymines at the junction of 19mer/ 46merWCfH complex increased Tm by only +5 °C with respect to the 17mer/46merWCfH. The presence of one additional thymine in the 19mer/46merWCfH complex might slightly distort the structure of the 18mer/ 46merWCfH complex (Table 1). The introduction of an acridine derivative at the junction between the triple helix and the double helix domains of the 17mer/46merWCfH complex increased the binding affinity by +16 °C (Table 1). The Tm of the 17mer/46merWCfH and 17mer-X/46merWCfH complexes are, respectively, 34 and 50 °C in the presence of 0.2 mM spermine (Figure 7A). It should be noted that the acridine at the junction (in 17mer-X/46merWCfH complex) increases the binding affinity much more than one or two thymines at the junction (in 18mer/ 46merWCfH and 19mer/46merWCfH complex), demonstrating an important stabilizing role of the intercalating agent at the junction of WCfH structures. We have also measured the effect of covalent attachment of the acridine derivative to the 5′ end of the 17mer ligand. Acr-17mer binds to the WCfH complex forming a double helix with the 5′ sequence and a triple helix with the stem of the 46merWCfH (Figure 7). The acridine can intercalate in the stem sequence at the junction between the triple helical and the double helical domains as previously demonstrated for triplex-forming acridineoligopyrimidine conjugates (39). The presence of the intercalating agent at the 5′ end of the 17mer (Acr-17mer) increased the Tm by +15 °C (Figure 7 and Table 1), a value close to that obtained with the acridine attached

Figure 7. Schematic representations of the complexes formed by the hairpin containing targets (T) 46mer WCfH with the acridine-containing or unmodified oligonucleotide ligands (L). Melting temperatures indicated below each complex were obtained with 2 µM/2 µM T/L in a pH 7.02 cacodylate buffer (50 mM) containing 0.1 M NaCl and either 0.2 mM (A) or 1 mM spermine (B). ∆Tm values are calculated as the difference between the Tm values of the acridine-containing and the corresponding unmodified oligonucleotide.

to the internal site. Surprisingly, the bis-modified oligonucleotide Acr-17mer-X did not exhibit a better affinity for the target 46merWCfH than the acridine-containing oligonucleotides, 17mer-X and Acr-17mer, a similar result to that obtained in the first model of a hairpin target described in this paper (model 1). To obtain more information on the type of interactions engaged by the internally located acridine, an additional thymine was inserted either between the duplex-forming sequence and the acridine (18mer-XT) or between the acridine and the triplex-forming sequence (18mer-TX). In both cases, the Tm (50 °C) was identical to that of the 17mer-X/46merWCfH complex (only 18mer-TX is shown in Figure 7 and Table 1). As compared to the 18mer, which contains the additional thymine but no acridine, the stabilization afforded by the acridine is only +8 °C as compared to +16 °C for the 17mer. These results demonstrates that two elements contribute to the acridine-induced stabilization: an increased flexibility at the junction between the two domains of the 17mer and a specific interaction of the acridine ring at the junction of the duplex and triplex structures. Spermine is known to stabilize triple helices (40, 39). When the spermine concentration was raised from 0.2 to 1 mM, the Tm of the 17mer was increased by 3 °C (from 34 to 37 °C). Under these conditions, the lack of a thymine in the triplex-forming sequence of the 16mer decreased the Tm value by 2.5 °C (Figure 7B). Introduction of the intercalating agent in 16mer-X stabilized the interaction with the 46merWCfH target [∆Tm(16mer-X/16mer) ) +14.5 °C] (Figure 7B). A similar stabilization was observed with the 5′-substituted Acr-16mer.


Figure 8. First derivative profiles of the melting curves obtained at 260 nm for 2 µM 46merWCfH bound to 2 µM of 17mer (b), 17mer-X (O), Acr-17mer (4), 18mer (9), and 18merTX ([) in 50 mM cacodylate buffer, pH 7.02, containing 0.1 M NaCl.

Melting experiments with oligonucleotide ligands and the 46merWCfH target were also performed in the absence of spermine. Melting profiles obtained in the absence of spermine with 17mer, 17mer-X, and Acr17mer bound to the 46merWCfH (Figure 8) demonstrated that the acridine ring better stabilized the complexes when it was located at the internal position than at the 5′ end of the 17mer oligonucleotide [∆Tm(17mer-X/17mer) ) +21 °C, ∆Tm(Acr-17mer/17mer) ) +17.5 °C]. A similar behavior was observed with the 18mer and 18mer-TX (Table 1). In the 17mer/46merWCfH complex in the absence of spermine, the triplex-forming portion of the 17mer (the 5′ sequence) was not expected to form a stable triple helix with the stem sequence of the 46merWCfH. As a matter of fact, mutations introduced in this part of the 17mer sequence (as in 17mer-Mut) did not markedly change the Tm value of the complex (∆Tm ) -1.5 °C). For the Acr17mer oligonucleotide, it is likely that a triplex is formed between the 5′ sequence of the 17mer and the stem sequence of the 46mer WCfH with the acridine intercalated at the junction of triplex and duplex domains (39). The increase in Tm value obtained when the 17mer is 5′substituted with acridine (Acr-17mer, ∆Tm ) +17.5 °C) and when it is internally substituted (17mer-X, ∆Tm ) +21 °C) strongly suggests that 17mer-X also forms a complex involving the triplex portion even in the absence of spermine. The internal acridine could be either intercalated at the junction or located at a preferential binding site within the branch point of the WCfH structure leading to a high stabilization of complexes. CONCLUSION

In the present study, we demonstrated that an acridine incorporated at an internal site within an oligonucleotide is able to stabilize the binding of this oligonucleotide to a hairpin-containing target sequence when the acridine ring is located at the base of the stem-loop region (model 1). The internal acridine leads to a better stabilization of complexes (∆Tm ) +13 °C) than an acridine covalently linked to the 5′-end of the oligonucleotide (∆Tm ) +8 °C), suggesting the existence of a preferential site of interaction/intercalation for the acridine ring at the three-way junction. The binding of oligonucleotides to hairpincontaining nucleic acids could also be stabilized by in-


sertion of modified bases such as a 5-methyl-N4-(1pyrenylmethyl)cytidine or a 2′-deoxy-5-methyl-N4-(4-phenoxyphenyl)cytidine instead of an acridine ring (41-43). Acridine-containing oligonucleotides have also been targeted to hairpin-containing targets in such a way as to form both Watson-Crick base pairs and Hoogsteen base pairs (WCfH recognition, model 2). In the presence of spermine, no differences in stabilization of WCfH complexes were observed between an internal acridine and an acridine linked to the 5′ extremity of the oligonucleotide. Melting temperatures of DNA complexes were increased by +15-16 °C. In the absence of spermine, the oligonucleotide with an internal acridine bound to the hairpin-containing target exhibited a higher stability than the 5′ acridine-oligonucleotide conjugate (∆Tm ) +21 °C compared to +17.5 °C). What has emerged from our study is that acridine incorporated into an oligonucleotide could be used to recognize hairpin-containing nucleic acids with higher affinity than the unmodified oligonucleotides either by formation of a three-way structure or by a combination of double and triple helix formation. These oligonucleotide-intercalator conjugates are easily synthesized with existing building blocks and provide a new approach to selectively recognize secondary structures in nucleic acids. Their potential applications in the antisense strategy should be evaluated in future investigations. ACKNOWLEDGMENT

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BC9801225

Recognition of Hairpin-Containing Single-Stranded DNA by

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