Stem–loop oligonucleotides as tools for labelling double-stranded DNA Be ´ ne ´ dicte Ge ´ ron-Landre, Thibaut Roulon and Christophe Escude ´ Laboratoire ‘Re ´ gulation et Dynamique des Ge ´ nomes’, De ´ partement ‘Re ´ gulations, De ´ veloppement et Diversite ´ Mole ´ culaire’, Muse ´ um National d’Histoire Naturelle, Paris Triple-helix forming oligonucleotides (TFOs) repre- sent an interesting tool for the sequence-specific recognition of double-stranded DNA. They can be used for the artificial modulation of DNA informa- tion processing [1] and for other applications that take place in vitro, such as double-stranded DNA isolation, labelling or modification (reviewed in [2]). Formation of DNA triple helices has been studied in details for the past 15 years (reviewed in [3]). Two different motifs of DNA triple helices can be formed, depending on the base composition of the TFO. Binding of the TFO occurs at oligopurineÆ oligopyrimidine sequences. Pyrimidine-rich oligonucleo- tides bind with a parallel orientation with respect to the oligopurine strand, by forming TÆAxT and CÆGxC + base triplets, whereas purine-rich oligonucleo- tides bind with an antiparallel orientation by forma- tion of TÆAxT, TÆAxA or CÆGxG base triplets. The conditions that favour triple-helix formation have been well characterized. The pyrimidine motif is usu- ally more stable at acidic pH, due to the require- ment for cytosine protonation, whereas very stable triple helices can be formed within the purine motif at neutral pH, provided the target sequence contains a high proportion of CÆG pairs and dications are present. G-rich oligonucleotides often fold into G-tetrad containing structures that can compete with triple-helix formation, thereby limiting in practice the use of this type of triple helix. Various strategies have been developed that permit the recognition of mixed sequence duplex DNA targets at physiological pH [4,5]. Keywords triple helix; DNA labeling; stem–loop oligonucleotide; sequence specificity; padlock oligonucleotide Correspondence C. Escude ´ , Laboratoire ‘Re ´ gulation et Dynamique des Ge ´ nomes’, De ´ partement ‘Re ´ gulations, De ´ veloppement et Diversite ´ Mole ´ culaire’, USM 0503 Muse ´ um National d’Histoire Naturelle, CNRS UMR5153, INSERM U565, Case Postale 26, 43 rue Cuvier, F-75231 Paris Cedex 05, France Fax: +33 14079 3705 Tel: +33 14079 3774 E-mail: escude@mnhn.fr (Received 23 June 2005, revised 17 August 2005, accepted 23 August 2005) doi:10.1111/j.1742-4658.2005.04932.x We report on a sequence-specific double-stranded DNA labelling strategy in which a stem–loop triplex forming oligonucleotide (TFO) is able to encircle its DNA target. Ligation of this TFO to either a short hairpin oligonucleotide or a long double-stranded DNA fragment leads to the for- mation of a topological complex. This process requires the hybridization of both extremities of the TFO to each other on a few base pairs. The effects of different factors on the formation of these complexes have been investi- gated. Efficient complex formation was observed using both GT or TC TFOs. The stem–loop structure enhances the specificity of the complex. The topologically linked TFO remains associated with its target even under conditions that do not favour triple-helix formation. This approach is suffi- ciently sensitive for detection of a 20-bp target sequence at the subfemto- molar level. This study provides new insights into the mechanics and properties of stem–loop TFOs and their complexes with double-stranded DNA targets. It emphasizes the interest of such molecules in the develop- ment of new tools for the specific labelling of short DNA sequences. Abbreviations BQQ, (6-[3-(dimethylamino)propyl]amino-11-methoxy-benzo[f]quino-[3,4-b]quinoxaline); TFO, triplex forming oligonucleotide. FEBS Journal 272 (2005) 5343–5352 ª 2005 FEBS 5343 For in vitro applications, the experimental conditions can be chosen to favour the formation of very stable tri- ple helices. Nevertheless, it may be desirable to form complexes that will resist extensive washing or an important dilution. TFOs can be covalently linked to their target by photocrosslinking of psoralen–oligo- nucleotide conjugates [6,7] or by using oligonucleotides conjugated to alkylating agents such as chlorambucyl [8]. We have described a triple-helical complex in which the third strand is topologically linked to its target [9]. This was achieved by circularization of the TFO after it had wound around its double-stranded DNA target thanks to triple-helix formation. When the target was carried by a circular DNA, i.e. a plasmid, the TFO was irreversibly linked to the plasmid. We have shown that the stability of the triple helix made by the topologically linked TFO, also called padlock oligonucleotide, was enhanced compared to that formed with a linear TFO [10]. For example, we showed that such a padlock oligo- nucleotide strongly inhibits DNA digestion by a restric- tion endonuclease, and that the complex is strong enough to inhibit the elongation of transcription by an RNA polymerase [11]. The triple helix used in these studies involved a third strand containing G and T, binding of which was stabilized by the use of a triplex specific intercalator. This made possible the use of a third strand which did not contain many Gs. Moreover, as the triple helix was not stable in the absence of inter- calator, it was possible to switch easily from conditions where the triple helix was very stable to conditions where it was totally unstable. A derivative of this approach was developed in which the ends of the TFO hybridized to each other and were ligated to either a short stem–loop oligonucleotide or to a DNA fragment that had a complementary sticky end (Fig. 1). The formation of these structures may be used for the pur- pose of grafting ligands to double-stranded DNA [12] or for visualizing short double-stranded sequences by fluor- escence microscopy [13]. Both structures were formed only if the samples were heated before the ligation reaction was started. Our idea was that the stem could dissociate upon heating, thereby allowing triple-helix formation between the loop of the TFO and the target during the cooling step, before rehybridization of the complementary sequences in the stem can take place. A stem length of 6 or 8 bp had been arbitrarily chosen in both studies. Although the topological link has been clearly established for both structures, no detailed study regarding the influence of stem length and heating con- ditions had been performed. The aim of the present study was to investigate the features of this type of complex. Efficient complex for- mation was observed using both the pyrimidine and purine motif triple helices. In particular, the influence of heating conditions and of the length of the stem has been studied as well as the specificity and the sensitiv- ity of this approach for DNA detection. Results Oligonucleotide design and labelling strategy We chose as a target sequence for the present study a 20-bp oligopurineÆoligopyrimidine sequence that is re- presented only once in the yeast genome (D. Polverari and J.S. Sun, personal communication). We designed two types of TFO with a stem–loop structure that can form a triple helix by binding to this sequence (Fig. 2). The first type contains G and T in the loop of the AB Fig. 1. Scheme of the padlock structures. The central part of the TFO forms a triplex with the target dsDNA. The 5¢-and3¢-part of the TFO hybridize to each other, thereby forming a short double- stranded stem, and leaving a four nucleotide single-stranded dan- gling end. This end hybridizes to the complementary extremity of either a short hairpin oligonucleotide (A) or a long DNA fragment (B). A ligation reaction results in the formation of a closed dumbell- like oligonucleotide or a very long stem–loop structure. In both cases, the TFO encircles the target. Rupture of the topological link requires cleavage of the circular oligonucleotide (A) or denaturation of the double-stranded DNA (B). Fig. 2. Sequence of targets and TFOs used in this study. The cen- tral part of TG and TC TFOs as well as the target sequences inser- ted in plasmids pY, pY1m and pY2m are shown. The pY plasmid contains a 20-bp oligopurineÆoligopyrimidine target sequence, shown in bold. The pY1m and pY2m plasmids have the same target sequence as pY except for one or two mismatch(es), respectively, which are shown underscored. This sequence can form a triplex made of TÆAxT and CÆGxC + triplets with a parallel TC TFO or a triplex made of TÆAxT and CÆGxG triplets with an antiparallel TG TFO. Stem–loop padlock oligonucleotides for dsDNA B. Ge ´ ron-Landre et al. 5344 FEBS Journal 272 (2005) 5343–5352 ª 2005 FEBS TFO and can form a triple helix in the presence of the triplex-stabilizing agent 6-[3-(dimethylamino)propyl]- amino-11-methoxy-benzo[f]quino-[3,4-b]quinoxaline (BQQ), as already described in our previous work [10–14]. The second one can form a triple helix at acidic pH. The 3¢- and 5¢-regions of the TFO can hybridize to each other, thereby forming a short double-stranded stem of variable length and leaving a 4-base single-stranded dangling 5¢-end, which can be ligated using T4 DNA ligase to any DNA with a com- plementary sticky end. The sequences of the TFOs used in this study are summarized in Table 1. These TFOs can be ligated to either a short hairpin oligo- nucleotide or a DNA fragment with a 4-bases sticky end (Fig. 1). In both cases, ligation in the presence of the double-stranded target DNA may result in the for- mation of a topologically linked complex, which we call a padlock. We checked by denaturing PAGE that all the stem–loop TFOs could be efficiently ligated to the hairpin oligonucleotide under the experimental conditions used in the absence of the target DNA, irrespective of the length of the stem (data not shown). Padlock formation with GT oligonucleotides We first studied the formation of topologically linked complexes using GT TFOs and the short hairpin oligo- nucleotide. The TFO was radiolabelled and used in excess over the amount of target. After the ligation reaction, samples were analysed by agarose gel electro- phoresis and autoradiographed. Two bands are observed, corresponding to the supercoiled and relaxed forms of the plasmid. The labelling yield can be esti- mated from the amount of labelled oligonucleotide that comigrates with the plasmid. No labelling was observed when the samples were not heated (Fig. 3B, lane 4) or when ligase was omitted (data not shown). The length of the stem was varied from 6 to 11 bp (see Table 1 for sequences). The maximal yields were achieved for lengths between 8 and 10 bp (Fig. 3A, lanes 1–4). We tried to vary the cooling rate between 80 °C and 30 °C. The best yields were achieved at a rate of 0.25 °CÆmin )1 (Fig. 3B). A cooling rate of 0.25 °CÆmin )1 was therefore preferred for subsequent experiments with GT TFOs. Padlock formation with TC oligonucleotides The use of TC TFOs has not been previously described for the formation of topologically linked complexes where a TFO encircles its double-stranded DNA tar- get. Our previous attempts came up with the fact that formation of the triple helix requires a pH that is too low to enable efficient ligation by T4 DNA ligase. To circumvent this problem we decided to perform the incubation step at acidic pH, and then to neutralize the sample before addition of T4 DNA ligase. An aci- dic pH should favour triplex formation without affect- ing the stability of the stem. After cooling the sample, the stem–loop structure should remain stable upon increasing the pH. Therefore, addition of the ligase should result in the formation of the topological link even if the triple helical structure has become unstable. Such a protocol, carried out by forming the triple helix at pH 5 and neutralizing to pH 7.5, led indeed to the efficient formation of a topologically linked complex using various TC TFO (Fig. 3). The influence of stem length and heating conditions were also investigated in this case. The greatest yields were obtained for a stem length of 8 bp (Fig. 3A, lanes 5–7). The yield of com- plex formation was lower when the samples were hea- ted to a maximal temperature lower than 80 °C (data not shown) or when the samples were not heated (Fig. 3B, lane 8). We tried to vary the cooling rate between 80 °C and 30 °C. The best yields were achieved at a rate of Table 1. TFOs used in this study. The name and sequence of the TFOs are indicated. The TC TFO (TC4, TC6, TC8 and TC10) have a central sequence made of T and C. The TG TFO (TG6, TG8, TG10 and TG11) have a central sequence made of T and G. The central sequences that recognize the target sequence by triple-helix formation are represented in bold. On both sides, the sequences that hybridize to each other in order to form a double-helical stem are underlined whereas the single-stranded sticky ends are shown in italics. TFO Sequence TC4 CGGTCCTATTTCGACGCTAGCTTTTTTTTCTCTTTCCTCCTTTTCTTTTCACGTGGAGCTTCTAGG TC6 CGGTCCTAGTTCGACGCTAGCTTTTTTTTCTCTTTCCTCCTTTTCTTTTCACGTGGAGCTACTAGG TC8 CGGTCCTAGTACTCGACGCTAGCTTTTTTTTCTCTTTCCTCCTTTTCTTTTCACGTGGAGCTGTACTAGG TC10 CGGTCCTAGTACGCTCGACGCTAGCAAAATTTTCTCTTTCCTCCTTTTCAAAACACGTGGAGCTGCGTACTAGG TG6 CGGTCCTAGTTCGACGCTAGCAAAAGTTTTGGTGGTTTGTGTTTTAAAACACGTGGAGCTACTAGG TG8 CGGTCCTAGTACTCGACGCTAGCAAAAGTTTTGGTGGTTTGTGTTTTAAAACACGTGGAGCTGTACTAGG TG10 CGGTCCTAGTACGCTCGACGCTAGCAAAAGTTTTGGTGGTTTGTGTTTTAAAACACGTGGAGCTGCGTACTAGG TG11 CGGTCCTAGCTACGCTCGACGCTAGCAAAAGTTTTGGTGGTTTGTGTTTTAAAACACGTGGAGCTGCGTAGCTAGG B. Ge ´ ron-Landre et al. Stem–loop padlock oligonucleotides for dsDNA FEBS Journal 272 (2005) 5343–5352 ª 2005 FEBS 5345 0.25 °CÆmin )1 (Fig. 3B), as observed for the GT oligo- nucleotides. However, we noticed that a long heating in the acidic buffer resulted in plasmid nicking. There- fore, a cooling rate of 1 °CÆmin )1 was prefered for subsequent experiments with TC TFOs in order to preserve the supercoiled conformation of the target plasmid. Specificity of padlock formation Triple-helix formation is a sequence specific process and it has been reported that the presence of mis- matches between the third strand and the target duplex decreases the stability of triple helical complexes [15,16]. We wondered whether the use of stem–loop TFOs would affect the specificity of com- plex formation. We therefore constructed two other plasmids (pY1m and pY2m) containing the 20-bp oligopurineÆoligopyrimidine target sequence where 1 or 2 mismatches have been introduced (Fig. 2). For- mation of the topological complex was carried out in the presence of equimolar amounts of the two dif- ferent plasmids (i.e. pY and pY1m or pY and pY2m) in order to study the specificity of complex formation. Labelling of the pY2m plasmid was never observed (data not shown). Labelling of pY1m was observed with a much lower yield than for pY (Fig. 4). The labelling ratio between the two plas- mids was used to estimate the specificity. This ratio increased from 5 to 8 when the stem length of a TC TFO increased from 6 to 10 bp and from 9 to 14 when the stem length of a TG TFO increased from 6 to 11 bp (Fig. 4). Sensitivity of detection with a radiolabelled padlock Radioactive labelling is commonly used for sensitive detection of DNA. Enzymatic synthesis of a DNA fragment in the presence of a radiolabelled nucleotide results in the incorporation of more isotopic labels at the target site than direct phosphorylation of a TFO. Therefore, we investigated the sensitivity of a DNA detection assay based on ligation of a radiolabelled DNA fragment to a TFO. [ 32 P]dCTP[aP] was incor- porated into a 0.5 kb DNA fragment during PCR. Fig. 4. Specificity of padlock formation. Padlocks were formed in a mix containing 100 ng (5 n M) of pY (3.0 kb) and 170 ng (5 nM)of pY1m (4.4 kb). Controls were performed with pY alone (lanes 1 and 7) or pY1m alone (lanes 2 and 8). Padlocks were formed with radiolabelled GT TFOs (lanes 1–6) or TC TFOs (lanes 7–11) and the short hairpin oligonucleotide. These TFOs differ in the length of the double-stranded stem, as indicated by the number in their names. The relative rate of padlock formation for the perfectly matched and mismatched sequences is shown below the gels. A B Fig. 3. Yields of padlock formation. (A) Influence of triple-helix motif and stem length. (B) Influence of the cooling rate. Padlocks were formed on the pY plasmid using radiolabelled GT TFOs (lanes 1–4) or TC TFOs (lanes 5–7) and the hairpin oligonucleotide. The TFOs differ in the length of the double-stranded stem, as indicated by the number in their names. During padlock formation, the samples were heated to 80 °C and cooled to 30 °C at various rates, as indi- cated. A minus sign (–) indicates that the samples were not heated at all (lanes 4 and 8). After the ligation reaction, complexes were analysed on an agarose gel, which was dried and autoradiographed. The intensities of both bands were quantified and summed. The yield of padlock formation is shown below the gels, relatively to the best yield for each experiment. Stem–loop padlock oligonucleotides for dsDNA B. Ge ´ ron-Landre et al. 5346 FEBS Journal 272 (2005) 5343–5352 ª 2005 FEBS The pY plasmid was incubated in the presence of the nonlabelled 5¢-phosphorylated TFO (20 nm) and the radiolabelled fragment (30 nm), and the sample was heated before addition of T4 DNA ligase. After migra- tion of the samples in a 1% agarose gel, comigration of the labelled fragment and the plasmid was observed. We were able to detect 250 attomol of plasmid without ambiguity, and a band could be identified with as few as 50 attomol (Fig. 5). Padlock stability The stability of the triple helical complex relies on the presence of the triplex stabilizing agent BQQ or on acidic pH. Therefore, stability may become a lim- iting factor when further experiments are conducted in the absence of BQQ or at neutral pH. On the other hand, the topological link provides enhanced stability to the complex. In order to study the mobility of the topologically linked circular TFOs, the pY plasmid was first linearized using different restriction endonucleases before padlock formation. The restriction sites were located at three different positions located between 74 and 1040 bp away from the target site (Fig. 6). The linearized DNA mole- cules were still labelled after migration in a 1% agarose gel (Fig. 6, lanes 2–4 and 6–8), indicating that the topologically linked complexes are stable enough not to dissociate during gel electrophoresis. The migration rate of the modified linear plasmids was observed to vary with different restriction enzymes, which suggests that the migration rate depends on the position of the label on the linear molecule. The slowest migrations were obtained when the attachment site was located close to the middle of the linear DNA. The sharpness of the different bands and the different migration rates suggest that the short DNA fragment remains tightly associated with its target sequence during electrophoresis. Discussion The aim of this work was to investigate the formation of topological complexes where a stem–loop oligo- nucleotide encircles a double-stranded DNA molecule. Formation of these structures uses triple-helix forma- tion to wind an oligonucleotide around a double-stran- ded DNA target. The TFO is then ligated to a short hairpin oligonucleotide or to a longer double-stranded DNA, which results in a topological link between the TFO and the target duplex. The influence of various parameters such as TFO stem length and heating tem- perature have been studied, as well as the sensitivity and specificity of this approach. Heating the samples is necessary for efficient complex formation. Stem length and heating conditions have a great influence on the labelling yield for both GT and TC TFOs. An optimal Fig. 5. Plasmid detection with a radiolabelled DNA fragment. A radio- labelled DNA fragment was ligated to a TFO (TG8) in the presence of different amounts of the pY plasmid. The quantity of plasmid is indi- cated, as well as the signal to noise ratio (S ⁄ N). Fig. 6. Padlock stability on linear DNA. Plasmid pY was digested with the restriction enzymes XmnI, DraIII, or XbaI, which cut at 1040, 498, and 74 bp, respectively, from the triplex target site. Pad- locks were formed on the undigested plasmid (lanes 1 and 5) or on the linearized plasmids (lanes 2–4 and 6–8) with the TFO TG8 (lanes 1–4) or TC8 (lanes 5–8) and a radiolabelled DNA fragment. The small band in lane 6 which migrates like the main band in lane 8 may be due to some radiolabelled fragment ligated at the end of the linearized plasmid. B. Ge ´ ron-Landre et al. Stem–loop padlock oligonucleotides for dsDNA FEBS Journal 272 (2005) 5343–5352 ª 2005 FEBS 5347 labelling yield is obtained when the samples are heated up to 80 °C before the temperature is slowly decreased, and for intermediate stem length values. For the first time, we demonstrate that topologically linked complexes can be assembled on supercoiled plasmids using TC TFOs, which bind double-stranded DNA at acidic pH, in place of GT TFOs that bind in the presence of triplex stabilizing agents. This was possible by performing the hybridization and ligation steps at different pHs. This allows the process to be conducted in the absence of any DNA intercalating agents. A topologically linked complex involving a TC oligonucleotide had been previously described [17]. In this study, a precircularized TFO was threaded on a short double-stranded DNA before this short double- stranded DNA was ligated into a longer circular, non- supercoiled molecule. This strategy cannot be used for labelling circular DNA. In our approach, TFOs do not have to thread on the target DNA. They are probably not able to thread on long DNA anyway, as shown by experiments with phage k DNA [13]. A heating step is necessary to open the stem–loop structure, which can reassociate after triple-helix formation (Fig. 7). We have previously sug- gested that in the absence of heating, the TFO becomes ligated while it is not wound around the dou- ble-stranded DNA target. Whether this circular TFO can still form a triple helical structure after ligation remains an unresolved question, as the formation of triple helices by linear TFOs could not be detected by gel electrophoresis under our experimental conditions. But the length of the linkers between the region that forms a triple helix and the stem (14 nucleotides on both sides) is probably too short to permit binding of the ligated TFO to more than 10 base pairs of the target (i.e. one turn around the double helix). In contrast, the topological link provides an enhanced stability, which results in a band shift that can be clearly detected. The proposed model can be further exploited in order to explain the results observed in the present study (Fig. 7). For long stem lengths, the stem can dissociate only at very high temperatures and is likely to reassociate at a temperature higher than the one at which triplex formation occurs. The ligation reaction will result in a complex that does not encircle the tar- get DNA. The decreased efficiency observed with very short stem lengths requires a different explanation. The short double-helical structure has a low stability. Therefore, hybridization of the short single-stranded ends may be inhibited by the tension exerted on them by the triple helical structure. We observed indeed that ligation was inhibited in the presence of an excess of plasmid (not shown). This hybridization may occur when the TFO dissociates from its target, resulting in a complex that does not encircle the double helix tar- get after ligation. Therefore, the ligation reaction drives the complex towards circularization outside of the double-stranded target. The effects of the heating conditions on labelling efficiency can probably also be explained by this model, taking into account the relat- ive kinetics of both triplex and stem–loop formation. A high cooling rate may not be compatible with effi- cient triple-helix formation due to slow association kinetics [18]. Therefore the TFO stem might reassoci- ate while the triple-helix has not formed, resulting in a complex that does not encircle the target DNA. We have shown that a longer stem provides an enhanced specificity to the labelling reaction. This observation can be explained by noticing that the pres- ence of mismatches will decrease the melting tempera- ture of the triple-helical complex, which may become lower than the melting temperature of the stem. Other Fig. 7. Scheme for interpretation of the results. The target, the stem–loop TFO and the short hairpin oligonucleotide are represen- ted. The padlock structure (centre) forms upon dissociation of the stem (1) followed by triple-helix formation (2), stem reformation (3) and ligation of the hairpin oligonucleotide (4). When the stem is lon- ger, ligation of the TFO may occur outside of the target before tri- ple-helix formation (right). When the stem is too short (left), the formation of a triple helical complex may inhibit stem reassociation. The reassociation and ligation may happen while the triple-helix is dissociated. Stem–loop padlock oligonucleotides for dsDNA B. Ge ´ ron-Landre et al. 5348 FEBS Journal 272 (2005) 5343–5352 ª 2005 FEBS strategies have been described for designing DNA probes with an enhanced specificity. For example, molecular beacons are stem–loop oligonucleotides which are used in a specific single-stranded DNA detection assay based on fluorescence quenching [19,20]. Competition between formation of the stem– loop structure and hybridization to the target results in an increased specificity. The stem–loop oligonucleo- tides presented in this paper differ from molecular bea- cons as the stem–loop structure and the triple-helical complex exist simultaneously, in contrast to molecular beacons. Formation of the stem–loop structure reinfor- ces the strength and specificity of the triple-helical structure. Several oligopurineÆoligopyrimidine sequences have been used as targets for attaching stem–loop padlock oligonucleotides ([12,13], B. Ge ´ ron-Landre, T. Roulon, M. Bello-Roufaı ¨ & C. Escude ´ , unpublished results). Their length varies from 12 to 20 base pairs. We present here the first systematic study of stem length for a 20-bp target sequence and two derivatives with one or two mis- matches. The yields have been shown to depend on the length of the stem. More generally, they are likely to depend on the stability of both the stem and the triple helix, i.e. on their length and base pair composition. Formation of the catenated complex is sequence specific. This feature makes our approach an interesting one for labelling genomic targets. Experiments with phage k DNA have confirmed this trend [13], and the present results will help in the design of experiments aimed at targeting a single sequence within the yeast Saccharo- myces cerevisiae genome. OligopurineÆoligopyrimidine sequences are over-represented in eukaryotic genomes [21], and strategies have been described which allow recognition of sequences containing single- or double- inversions as well as alternating oligopurineÆ.oligopyrim- idine sequences [22], which reinforce the interest of our labelling approach. We have also demonstrated that ligation of a radio- labelled DNA fragment to a stem–loop TFO can be used for sensitive sequence-specific detection of DNA. Other approaches have been proposed for this task, for example the use of strand-invading peptide nucleic acids oligonucleotides that facilitate binding of an oligonucleotide to a single-stranded target [23]. In this study, quantification of the target was achieved by extension of the hybridized oligonucleotide, which acts as a primer. In our approach, the label is present on the DNA fragment, and this reaction is independent from the target sequence. Padlock oligonucleotides for double-stranded DNA offer an interesting alternative to irreversible triplexes such as those obtained for example by irradiation of psoralen–TFO conjugates [6]. It is also possible to cir- cularize an oligonucleotide around a locally denatured DNA target. This can be achieved using peptide nucleic acids openers, forming a so-called earring com- plex that can be used for DNA labelling or isolation of specific sequences from genomic DNA [24]. In such complexes, it was believed that the fact that the circle was threaded between both DNA strands was required in order to inhibit sliding of the circular oligonucleo- tide. The present report shows that the circular TFO did not slide during gel electrophoresis even under con- ditions that were not favourable to triplex stability, such as the absence of the triplex stabilizing agent, or a pH that does not favour triple-helix formation. Pre- vious experiments, in which triplex formation competed with cleavage by a restriction enzyme, had shown that a circular TFO could remain tightly locked on its target sequence or leave the restriction site accessible, depend- ing on the presence of a triplex stabilizing agent [10]. Therefore, conditions that promote local mobility of the circular TFO are not sufficient to make it slide freely along the target DNA, and some forces must be exerted on the padlock oligonucleotide in order to make it move. Such forces may be provided by the movements of enzymes that translocate on DNA [25] or by processive enzymes like RNA polymerases [11]. In this regard, our system may provide an interesting tool for the study of protein movements on DNA. Sequence-specific DNA binding agents have several applications. For example, they can be used for grafting chemical moieties to plasmids, such as targeting peptides or fluorophores, in order to target plasmids towards spe- cific subcellular compartments [12] or to study their intracellular localization [26], respectively. This so-called ‘DNA vector chemistry’ is especially useful in gene ther- apy. Fluorescent detection of short oligopurineÆoligo- pyrimidine sequences on large genomic DNA molecules that have been stretched by microfluidic devices opens the way to new types of genomic studies [27]. The for- mation of catenated complexes has also been used for detection of single-stranded nucleic acid sequences by fluorescent in situ hybridization [28]. Interestingly, the topological link allows washing under stringent condi- tions. Similar detection technologies may be carried out with probes that recognize specific sequences on native DNA. In conclusion, we have studied the formation of topo- logically linked complexes between an oligonucleotide and a double-stranded DNA target. This versatile label- ling strategy uses two independent moieties: the first one is an oligonucleotide that recognizes a double-stranded DNA in a sequence specific way, the second one may be an oligonucleotide or a DNA fragment. The DNA B. Ge ´ ron-Landre et al. Stem–loop padlock oligonucleotides for dsDNA FEBS Journal 272 (2005) 5343–5352 ª 2005 FEBS 5349 fragment may be used for incorporation of multiple labels in order to enhance detection sensitivity. The stem–loop structure of the oligonucleotide displays unique and undescribed characteristics in terms of probe–target interactions which represent a new approach for enhancing the specificity of nucleic acid hybridization. Experimental procedures Oligonucleotides and chemicals Sequences of the TFOs are given in Table 1. The sequence of the short hairpin oligonucleotide was 5¢-ACCGTCCGG ATTGGCTTTTGCCAATCCGGA-3¢. This oligonucleotide was 5¢-phosphorylated during synthesis. The sequence of the primers used for PCR were 5¢-CGGTATCAGCTCACTC AAAG-3¢ (fw), and 5¢-ATGCTGGTCTCTACCGGCGAT AAGTCGTGTCTTAC-3¢ (rv). The rv primer was biotinyl- ated at the 5¢ end during synthesis. All these oligonucleotides were obtained from Eurogentec (Seraing, Belgium); their concentration was calculated using a nearest-neighbour model for absorption coefficients. TFOs were radiolabelled by incubating 10 pmol TFO in 20 lL T4 polynucleotide kinase buffer (New England Bio- labs, Beverly, MA, USA) with 10 lCi [ 32 P]ATP[cP] (Amer- sham, > 5000 CiÆmmol )1 ) and 5 U T4 polynucleotide kinase (New England Biolabs) for 1 h at 37 °C. Unincorporated [ 32 P]ATP[cP] was removed using Micro Bio-Spin columns (Bio-Rad, Hercules, CA, USA). For use with radiolabelled DNA fragments, TFOs were 5¢-phosphorylated by incuba- ting 300 pmol TFO in 50 lL T4 DNA ligase buffer (New England Biolabs) with 10 U T4 polynucleotide kinase for 2 h at 37 °C. Synthesis of the triplex stabilizing agent BQQ has been described previously [29]. Plasmids Plasmids pY, pY1m and pY2m were constructed by cloning the appropriate oligonucleotide pair between the HindIII and StyI sites of pBluescript SK+ (3.0 kb, Stratagene, La Jolla, CA, USA), between the HindIII and EcoRI sites of pBR322 (4.4 kb, New England Biolabs) and between the HindIII and EcoRI sites of pGL2 control (6.0 kb, Promega, Madison, WI, USA), respectively. The sequence of the target sequence inserted in these plasmids is indicated in Fig. 2. For experiments regarding the mobility of padlock oligo- nucleotides, 1 l g pY was linearized with 20 U of either XmnI (New England Biolabs), DraIII (New England Bio- labs) or XbaI (Amersham, Bucks, UK) in the recommended buffers for 3 h at 37 °C. In order to avoid recircularization or multimer formation, the digested plasmid was dephos- phorylated by adding shrimp alcaline phosphatase (USB, Cleveland, OH, USA) (1 U for XmnI, 12 U for DraIII, 2.5 U for XbaI) and incubating for 2 h at 37 °C. Phospha- tase was inactivated for 20 min at 65 °C. DNA was then ethanol precipitated and resuspended in Tris 10 mm pH 8.0. Preparation of radiolabelled DNA fragments PCR primers were designed in order to produce a fragment of a 0.5 kb starting from the pBluescript SK+ plasmid (Stratagene). The sequence of the fw primer was chosen in order to introduce a cleavage site for the BsaI restriction enzyme. The PCR was carried out by mixing in 50 lL Taq buffer (Promega) supplemented with 2 mm MgCl 2 1.6 lm of each primer, 200 lm of each dNTP, 50 lCi [ 32 P]dCTP[aP] (10 lCiÆ lL )1 , 3000 CiÆmmol )1 ) (Amersham), 10 pgÆlL )1 pBluescript and 0.1 UÆlL )1 Taq DNA poly- merase (Promega). After 30 cycles of 30 s at 94 °C, 30 s at 61 °C and 1 min at 72 °C, the concluding extension was carried out for 10 min at 72 °C. Primers and unincorporat- ed dNTP were removed using Qiagen (Valencia, CA, USA) PCR purification kits, using the standard protocol. Then the PCR products were digested overnight at 50 °C with 50 U of BsaI (New England Biolabs). The biotinylated extremities and the nondigested biotinylated PCR products were removed using streptavidin-coated magnetic beads (Dynabeads, Dynal, Oslo, Norway). The labelled fragments were then ethanol precipitated, resuspended in 10 mm Tris ⁄ HCl pH 8.0 and quantified on a gel by comparison with a standard. Padlock formation Unless otherwise stated, padlocks made of a TG TFO were formed with 20 nm of the TFO and either 30 nm of the hairpin oligonucleotide or the 0.5-kb DNA fragment incubated with 100 ng plasmid and 20 lm triplex stabil- izing ligand BQQ in 10 lL T4 DNA ligase buffer (50 mm Tris ⁄ HCl, 10 mm MgCl 2 ,10mm dithiothreitol, 1mm ATP, 25 lgÆmL )1 BSA, pH 7.8 at 25 °C). The sample was heated to 80 °C and cooled to 30 °Cata rate of 0.25 °CÆmin )1 in an MJResearch thermocycler. Forty units of T4 DNA ligase (New England Biolabs) were then added and the sample was incubated overnight at 20 °C. Padlocks made of a TC TFO were formed with 20 nm TFO and 30 nm of the hairpin oligonucleotide or the 0.5-kb DNA fragment incubated with 100 ng plasmid in 10 lL acetate buffer (20 mm ammonium acetate, 20 mm MgCl 2 ,3mm dithiothreitol and 1 mm ATP, pH 5.0 at 25 °C). The sample was heated to 80 °C and cooled down to 30 °C at a rate of 1 °CÆmin )1 in a MJResearch thermocycler. It was then cooled on ice and 2 lL of an ice-cold neutralizing solution (150 mm Tris pH 8.0, T4 DNA ligase 100 U ÆlL )1 ) were added. The sample was incubated overnight at 16 °C. Stem–loop padlock oligonucleotides for dsDNA B. Ge ´ ron-Landre et al. 5350 FEBS Journal 272 (2005) 5343–5352 ª 2005 FEBS Padlock formation was assessed by electrophoresis on a 1% agarose gel in 0.5· TBE buffer at room temperature. 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