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<!DOCTYPE article PUBLIC "-//NLM//DTD Journal Archiving and Interchange DTD v2.3 20070202//EN" "archivearticle.dtd">
<article xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" article-type="research-article"><?properties open_access?><front><journal-meta><journal-id journal-id-type="nlm-ta">Nucleic Acids Res</journal-id><journal-id journal-id-type="publisher-id">Nucleic Acids Research</journal-id><journal-title>Nucleic Acids Research</journal-title><issn pub-type="ppub">0305-1048</issn><issn pub-type="epub">1362-4962</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">16798913</article-id><article-id pub-id-type="pmc">PMC1500870</article-id><article-id pub-id-type="doi">10.1093/nar/gkl431</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Recombination R-triplex: H-bonds contribution to stability as revealed with minor base substitutions for adenine</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Shchyolkina</surname><given-names>Anna K.</given-names></name></contrib><contrib contrib-type="author"><name><surname>Kaluzhny</surname><given-names>Dmitry N.</given-names></name></contrib><contrib contrib-type="author"><name><surname>Arndt-Jovin</surname><given-names>Donna J.</given-names></name><xref rid="au1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Jovin</surname><given-names>Thomas M.</given-names></name><xref rid="au1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhurkin</surname><given-names>Victor B.</given-names></name><xref rid="au2" ref-type="aff">2</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><aff><institution>Engelhardt Institute of Molecular Biology, Russian Academy of Sciences</institution><addr-line>119991 Moscow, Russia</addr-line></aff><aff id="au1"><sup>1</sup><institution>Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry</institution><addr-line>D-37070 Goettingen, Germany</addr-line></aff><aff id="au2"><sup>2</sup><institution>Laboratory of Cell Biology, National Cancer Institute</institution><addr-line>NIH, Bethesda, MD 20892, USA</addr-line></aff></contrib-group><author-notes><corresp id="cor1"><sup>*</sup>To whom correspondence should be addressed. Tel: +1 301 496 8913; Fax: +1 301 402 4724; Email: <email>zhurkin@nih.gov</email></corresp></author-notes><!--For NAR: both ppub and collection dates generated for PMC processing 1/27/05 beck--><pub-date pub-type="collection"><year>2006</year></pub-date><pub-date pub-type="ppub"><year>2006</year></pub-date><pub-date pub-type="epub"><day>4</day><month>7</month><year>2006</year></pub-date><volume>34</volume><issue>11</issue><fpage>3239</fpage><lpage>3245</lpage><history><date date-type="received"><day>09</day><month>3</month><year>2006</year></date><date date-type="rev-recd"><day>29</day><month>5</month><year>2006</year></date><date date-type="accepted"><day>31</day><month>5</month><year>2006</year></date></history><copyright-statement>© 2006 The Author(s)</copyright-statement><copyright-year>2006</copyright-year><license license-type="openaccess"><p>This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/2.0/uk/"/>) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.</p></license><abstract><p>Several cellular processes involve alignment of three nucleic acids strands, in which the third strand (DNA or RNA) is identical and in a parallel orientation to one of the DNA duplex strands. Earlier, using 2-aminopurine as a fluorescent reporter base, we demonstrated that a self-folding oligonucleotide forms a recombination-like structure consistent with the R-triplex. Here, we extended this approach, placing the reporter 2-aminopurine either in the 5′- or 3′-strand. We obtained direct evidence that the 3′-strand forms a stable duplex with the complementary central strand, while the 5′-strand participates in non-Watson–Crick interactions. Substituting 2,6-diaminopurine or 7-deazaadenine for adenine, we tested and confirmed the proposed hydrogen bonding scheme of the A*(T·A) R-type triplet. The adenine substitutions expected to provide additional H-bonds led to triplex structures with increased stability, whereas the substitutions consistent with a decrease in the number of H-bonds destabilized the triplex. The triplex formation enthalpies and free energies exhibited linear dependences on the number of H-bonds predicted from the A*(T·A) triplet scheme. The enthalpy of the 10 nt long intramolecular triplex of −100 kJ·mol<sup>−1</sup> demonstrates that the R-triplex is relatively unstable and thus an ideal candidate for a transient intermediate in homologous recombination, t-loop formation at the mammalian telomere ends, and short RNA invasion into a duplex. On the other hand, the impact of a single H-bond, 18 kJ·mol<sup>−1</sup>, is high compared with the overall triplex formation enthalpy. The observed energy advantage of a ‘correct’ base in the third strand opposite the Watson–Crick base pair may be a powerful mechanism for securing selectivity of recognition between the single strand and the duplex.</p></abstract></article-meta></front><body><sec><title>INTRODUCTION</title><p>The alignment of three strands of nucleic acids, in which the third strand is identical and oriented parallel to one of the DNA duplex strands, occurs in recombination (<xref ref-type="bibr" rid="b1">1</xref>), at telomeres during t-loop formation (<xref ref-type="bibr" rid="b2">2</xref>), in DNA rearrangements caused by replication of mitochondrial DNA (<xref ref-type="bibr" rid="b3">3</xref>), and has been invoked in initiation of heterochromatin by small hairpin RNAs (<xref ref-type="bibr" rid="b4">4</xref>). In principle, such an alignment is capable of forming the R-triplex structure (recombination triplex DNA) as well as a strand exchange structure (D-loop) resulting from the third strand invasion into the duplex. An R-triplex (or R-form DNA) structure was predicted theoretically (<xref ref-type="bibr" rid="b5">5</xref>) to consist of isomorphic R-triplets; i.e. the putative R-triplex can accommodate any arbitrary sequence. R-type hydrogen bond schemes for the G*(C·G) and C*(G·C) triplets have been visualized by X-ray analysis (<xref ref-type="bibr" rid="b6">6</xref>,<xref ref-type="bibr" rid="b7">7</xref>), while the T*(A·T) and A*(T·A) triplets were verified by FTIR (<xref ref-type="bibr" rid="b8">8</xref>,<xref ref-type="bibr" rid="b9">9</xref>). Data supporting the existence of the R-triplex in protein-free systems derived from UV and fluorescence thermal denaturation curves, chemical probing, as well as gel shift and FRET assays (<xref ref-type="bibr" rid="b10">10</xref>–<xref ref-type="bibr" rid="b13">13</xref>) have been published by our laboratories.</p><p>Special oligonucleotide constructs (RCW fold) consisting of three strands connected with nucleotide linkers (<xref ref-type="fig" rid="fig1">Figure 1</xref>) in which the 5′-terminal strand is denoted as R (Recombination), the central strand as C (Crick, ‘complementary’) and the 3′-strand as W (Watson) have been used to study the alignment of the strands (<xref ref-type="bibr" rid="b13">13</xref>). The two linker loops differ in their conformational flexibility, the GAA loop being extremely stable, while the TTTT loop is less rigid. Importantly, such an RCW fold can accommodate any nucleotide sequence with the two identical R- and W-strands and a complementary C-strand.</p><p>We demonstrated previously that the fluorescent base analog, 2-aminopurine (2AP), can be substituted for adenine in the A*(T·A) triplet, forming the 2AP*(T·A) triplet, stereochemically consistent with the R-form (<xref ref-type="fig" rid="fig2">Figure 2</xref>). The temperature-dependent cooperative dissociation of the R-strand from the duplex was detected by two techniques: (i) fluorescence of the 2AP which monitored disruption of the individual A*(T·A) triplet and (ii) conventional UV absorbance at 260 nm reflecting melting of the third strand as a whole. Both methods revealed the same temperature-dependent cooperative dissociation of the R-strand from the duplex part (<xref ref-type="bibr" rid="b13">13</xref>), thus demonstrating that the fluorescence of the 2AP reported faithfully the thermodynamic parameters of the RCW triplex fold.</p><p>In the present work, we extended this approach by placing the fluorescent 2AP and other substitutions for adenine, site-specifically, either in the 5′- or 3′-strand. These substitutions allowed us (i) to monitor the difference in conformational behavior between the two identical R- and W-strands in the RCW fold and (ii) to obtain quantitative data on the energetics of the R-triplex formation. In particular, we were interested in deducing the relative impact of the base–base interactions in the duplex part of the triplex, and between the duplex and the third strand, i.e. issues directly related to the fidelity of nucleic acid recognition.</p></sec><sec sec-type="materials|methods"><title>MATERIALS AND METHODS</title><sec><title>Oligonucleotides</title><p>R<italic><sup>a</sup></italic>CW, RCW<italic><sup>a</sup></italic>, R<italic><sup>add</sup></italic>CW, R<italic><sup>a</sup></italic>CW<italic><sup>7</sup></italic>, R<italic><sup>a</sup></italic>CW<italic><sup>77</sup></italic>, R<italic><sup>aaa</sup></italic>CW<italic><sup>777</sup></italic> and CW<italic><sup>a</sup></italic> were synthesized and purified by high-performance liquid chromatography by Midland Certified Reagent Co. Inc. (TX); R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> and RCW<italic><sup>a</sup></italic> were synthesized and purified in PAAG by Syntol (Moscow) (for designations and folding schemes see <xref ref-type="fig" rid="fig1">Figure 1</xref>). The hydrogen bonding schemes for the A*(T·A) triplet and the triplets with 2AP, 2,6-diaminopurine (DAP) and 7-deazaadenine (7DAA) substitutions tested in the study are given in <xref ref-type="fig" rid="fig2">Figure 2</xref>. Samples contained 0.9–1.4 µM oligonucleotides, 0.5 M LiCl and 10 mM Tris–HCl buffer, pH 7.6.</p></sec><sec><title>Fluorescence measurements</title><p>The temperature dependence of the fluorescence emission of oligonucleotides containing a single 2AP substitution were recorded with a Cary Eclipse fluorescence spectrophotometer (Varian) in a thermostated cuvette at the constant heating of 0.5°C/min. The excitation wavelength was 310 nm, and the maximum of the emission was 370 nm. Samples contained 1 µM oligonucleotides, 0.5 M LiCl and 10 mM Tris–HCl buffer, pH 7.6.</p></sec><sec><title>UV thermal denaturation profiles</title><p>UV thermal denaturation curves were recorded at 260 nm with a Cary 100 Scan UV-visible spectrophotometer (Varian) with a constant heating gradient of 0.5 or 0.2°C/min.</p></sec><sec><title>Thermodynamic analysis of the intramolecular triplex formation</title><p>The detailed analysis of the thermal denaturation profiles, recorded by fluorescence of the 2AP reporter, has been described elsewhere (<xref ref-type="bibr" rid="b13">13</xref>). In essence, we analyzed the intramolecular binding of the dangling third strand to its double-helical CW part by fitting a theoretical curve to the experimental fluorescence melting curve. From these data we deduced the basic thermodynamic parameters of the intramolecular transition (denoted below as the ‘triple helix formation’).</p></sec></sec><sec><title>RESULTS AND DISCUSSION</title><sec><title>Probing conformation of the two homologous strands with 2AP</title><p>Substitution of 2AP for adenine constitutes an ideal structural probe for testing the RCW fold; the probe neither destabilizes nor distorts the 3D structure (<xref ref-type="bibr" rid="b13">13</xref>). Using 2AP as a reporter base in the 5′-strand, we demonstrated previously (<xref ref-type="bibr" rid="b13">13</xref>) that the R<italic><sup>a</sup></italic>CW oligonucleotide forms a sequence-specific structure, whose conformational equilibrium is shifted toward the R-type triplex. However, in these studies, we presented no direct evidence regarding conformation of the 3′-strand.</p><p>To detect the difference in conformation between the two identical R- and W-strands in the RCW fold, we substituted 2AP for adenine in the Watson 3′-strand (<xref ref-type="fig" rid="fig1">Figure 1</xref>, RCW<italic><sup>a</sup></italic> oligonucleotide) and compared the temperature dependence of the fluorescence intensity for RCW<italic><sup>a</sup></italic> with those for CW<italic><sup>a</sup></italic> and R<italic><sup>a</sup></italic>CW (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). The enhancement of R<italic><sup>a</sup></italic>CW fluorescence (circles) in the temperature range from 5 to 40°C reflected increasing exposure of 2AP to solvent due to melting of the triplex, namely a progressive loss of the 2AP stacking with the adjacent bases. Above 35–40°C, the R<italic><sup>a</sup></italic>CW melting profile followed that of the single R2AP strand (<xref ref-type="bibr" rid="b13">13</xref>). The temperature profile for the RCW<italic><sup>a</sup></italic> oligonucleotide was entirely different. In this case, the fluorescence monotonically decreased (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, squares), similar to the profile for the double-stranded hairpin CW<italic><sup>a</sup></italic> (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, diamonds). These results, and the fact that the RCW<italic><sup>a</sup></italic> and CW<italic><sup>a</sup></italic> oligonucleotides fluoresce similarly at equal concentrations, demonstrated that the fluorophore is in the same state in the RCW<italic><sup>a</sup></italic> and CW<italic><sup>a</sup></italic> folds, showing a temperature dependence of 2AP fluorescence that is typical for a DNA duplex. Thus, we conclude that the Watson 3′-strand retains base pairing with the complementary Crick strand under our experimental conditions.</p><p>The specific features of the melting profiles of 2AP fluorescence are likely related to a relatively hydrophobic environment with considerable base stacking inside the double helix (<xref ref-type="bibr" rid="b14">14</xref>) resulting in a markedly quenched fluorescence of the 2AP compared with that of 2AP in the third strand of the triplex. Further quenching of fluorescence of 2AP located within the double helix was probably promoted with increasing temperature through the non-radiative relaxation of excited states (<xref ref-type="bibr" rid="b15">15</xref>). A dynamic invasion of the 5′-strand in the Watson–Crick duplex displacing the identical 3′-strand appears improbable, as it would have caused a detectable increase in the 2AP fluorescence in RCW<italic><sup>a</sup></italic> compared with CW<italic><sup>a</sup></italic>. In other words, these data provide direct evidence against a dynamic mixture of two different duplex folds, i.e. branch migration structure, whereby the complementary C-strand is partially paired with both the 5′- and the 3′-strand.</p><p>Interpretation of the data is based on the assumption that the oligonucleotides form intramolecular folds rather than intermolecular associates. The nucleotide sequence, salt conditions and the procedure for the sample preparation have been selected to avoid formation of intermolecular species (<xref ref-type="bibr" rid="b13">13</xref>). The intramolecular character of the R<italic><sup>a</sup></italic>CW folding has been demonstrated by evaluating the oligonucleotide hydrodynamic volume under these experimental conditions (<xref ref-type="bibr" rid="b13">13</xref>). Here, we present additional, independent evidence for the intramolecular folding of R<italic><sup>a</sup></italic>CW by comparing melting curves for two concentrations of the oligonucleotide at 1 and 50 µM (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). The identity of two melting transitions for the concentrations differing by 50-fold unambiguously corroborates the intramolecular folding of the R<italic><sup>a</sup></italic>CW oligonucleotide.</p></sec><sec><title>2,6-Diaminopurine substitutions for adenines in the homologous R- and W-strands</title><p>To gain further information about the RCW fold structure, we made substitutions of adenines by DAP in the Watson and/or R-strands. The oligonucleotide R<italic><sup>add</sup></italic>CW contained two diaminopurines in the R-strand (<xref ref-type="fig" rid="fig1">Figure 1</xref>) in addition to the 2AP as in R<italic><sup>a</sup></italic>CW. Potentially, these additional substitutions would lead to formation of two DAP*(T·A) triplets (<xref ref-type="fig" rid="fig2">Figure 2</xref>), thereby stabilizing the RCW triplex through the additional H-bond in these triplets. Alternatively, the complementary C-strand may ‘change’ partners and form a canonical duplex with the 5′-R-strand (strand invasion), since DAP is known to pair with thymine forming three hydrogen bonds (<xref ref-type="bibr" rid="b16">16</xref>). We measured the temperature-dependent 2AP fluorescence of R<italic><sup>add</sup></italic>CW to distinguish between these two possibilities.</p><p>The temperature-dependent fluorescence curve of the R<italic><sup>add</sup></italic>CW oligonucleotide (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, triangles) gave a similar profile to that of CW<italic><sup>a</sup></italic> (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, diamonds) and RCW<italic><sup>a</sup></italic> (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, squares), but different from that of R<italic><sup>a</sup></italic>CW (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, circles). Based on these data, we conclude that the 5′-strand forms a duplex with the C-strand, which involves two stable T·DAP base pairs. Therefore, introduction of two DAP molecules in the 5′-strand strongly affects the mode of RCW folding and promotes strand exchange as opposed to an R-triplex. Note that the rearrangement of the RCW fold observed here upon the A substitutions by DAP in the R-strand is topologically equivalent to the strand exchange promoted by RecA protein (<xref ref-type="bibr" rid="b1">1</xref>,<xref ref-type="bibr" rid="b5">5</xref>).</p><p>In oligonucleotide R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic>, DAP was substituted for the adenines in both the 5′-R- and 3′-W-strands except for the reporter 2AP in the R-strand (<xref ref-type="fig" rid="fig1">Figure 1</xref>). According to the triplet scheme of <xref ref-type="fig" rid="fig2">Figure 2</xref>, we expected the two DAP*(T·DAP) triplets to stabilize the RCW fold by providing additional hydrogen bonds. Indeed, the R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> construct has a higher stability as follows from the fact that the temperature-dependent fluorescence profile is shifted rightward (<xref ref-type="fig" rid="fig4">Figure 4</xref>, circles).</p><p>Although DAP absorbs weakly at 310 nm, it was necessary to assess the potential contribution of the five DAP residues in the R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> construct to the R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> emission upon excitation at this wavelength. We compared the R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> fluorescence melting curve with the transition registered by UV absorption at 260 nm (<xref ref-type="fig" rid="fig4">Figure 4</xref>, inset, circles and solid line, respectively). The two curves coincide below 40°C, i.e. in the region where the UV melting reflects mainly the thermal dissociation of the third stand from the more stable duplex part of the R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic>. We conclude that the contribution of DAP fluorescence to the melting profiles of the oligonucleotide R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> (as well as R<italic><sup>add</sup></italic>CW, also containing the DAP bases) upon excitation at 310 nm is negligible.</p><p>Thermodynamic parameters for this construct as well as for R<italic><sup>a</sup></italic>CW, determined with our fitting procedure are listed in <xref ref-type="table" rid="tbl1">Table 1</xref>. The R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> triplex melting temperature increased by 6°C, resulting in gain in <italic>ΔH</italic> of ∼27 kJ·mol<sup>−1</sup> and an increase in <italic>ΔG</italic> of ∼4 kJ·mol<sup>−1</sup> at 0°C (<xref ref-type="table" rid="tbl1">Table 1</xref>). Thus, the R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> construct has the highest stability among the R-triplexes of mixed nucleotide sequences studied so far (<xref ref-type="bibr" rid="b10">10</xref>,<xref ref-type="bibr" rid="b11">11</xref>,<xref ref-type="bibr" rid="b13">13</xref>,<xref ref-type="bibr" rid="b17">17</xref>). The increased stability of the DAP*(T·DAP) triplets compared with the A*(T·A) triplets is consistent with the proposed H-bonding scheme for the R-triplet (<xref ref-type="bibr" rid="b5">5</xref>) (<xref ref-type="fig" rid="fig2">Figure 2</xref>).</p></sec><sec><title>Testing the H-bonding scheme of the A*(T·A) triplet with 7-deazaadenine substitutions</title><p>To further test the impact of hydrogen bonds on the stability of the R-triplex, we constructed oligonucleotides R<italic><sup>a</sup></italic>CW<italic><sup>7</sup></italic>, R<italic><sup>a</sup></italic>CW<italic><sup>77</sup></italic> and R<italic><sup>aaa</sup></italic>CW<italic><sup>777</sup></italic> with the 7-deazaadenine (7DAA) substituted for adenine in the Watson strand (<xref ref-type="fig" rid="fig1">Figure 1</xref>). In contrast to DAP, the 7DAA substitutions are expected to destabilize the RCW triplex (<xref ref-type="bibr" rid="b18">18</xref>). The 7DAA has a CH group in the ring position 7 instead of the negatively charged nitrogen N7. Hence, 7DAA cannot be an acceptor of a proton from the 2AP amino group and instead, the CH group would produce a steric clash with the amino group, thereby destabilizing the 2AP*(T·7DAA) triplet shown in <xref ref-type="fig" rid="fig2">Figure 2</xref>. Similarly, if an adenine is positioned in the 5′-R-strand opposite to 7DAA (see R<italic><sup>a</sup></italic>CW<italic><sup>77</sup></italic> in <xref ref-type="fig" rid="fig1">Figure 1</xref>), then the A–7DAA interaction is also expected to be unfavorable. In this case, the CH···HC electrostatic interaction (<xref ref-type="bibr" rid="b18">18</xref>) would be a major repulsion factor (<xref ref-type="fig" rid="fig2">Figure 2</xref>).</p><p>Thermal denaturation data for the R<italic><sup>a</sup></italic>CW<italic><sup>7</sup></italic> triplex are shown in <xref ref-type="fig" rid="fig4">Figure 4</xref> (triangles). R<italic><sup>a</sup></italic>CW<italic><sup>7</sup></italic> melted earlier than the R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> triplex by 7°C. Furthermore, the slopes of the melting curves were visibly different, corresponding to a significantly lower formation enthalpy and free energy for the R<italic><sup>a</sup></italic>CW<italic><sup>7</sup></italic> triplex (<xref ref-type="table" rid="tbl1">Table 1</xref>). Comparison of the thermodynamic parameters for the R<italic><sup>a</sup></italic>CW<italic><sup>7</sup></italic> triplex and the ‘reference’ R<italic><sup>a</sup></italic>CW fold indicates that the R<italic><sup>a</sup></italic>CW<italic><sup>7</sup></italic> is demonstrably less stable than the R<italic><sup>a</sup></italic>CW (<xref ref-type="table" rid="tbl1">Table 1</xref>), although the former differs from the latter by only one hydrogen bond (<xref ref-type="fig" rid="fig1">Figures 1</xref> and <xref ref-type="fig" rid="fig2">2</xref>).</p><p>The A*(T·A) triplet (<xref ref-type="fig" rid="fig2">Figure 2</xref>) is presumed to form a ‘weak’ CH···N hydrogen bond [reviewed in (<xref ref-type="bibr" rid="b19">19</xref>)]. To test the contribution of such a bond on R-triplex stability we constructed the oligonucleotide R<italic><sup>a</sup></italic>CW<italic><sup>77</sup></italic>, containing two putative A*(T·7DAA) triplets in addition to a ‘standard’ reporting 2AP*(T·A) triplet (<xref ref-type="fig" rid="fig1">Figure 1</xref>). This fold lacks two CH···N hydrogen bonds compared to the reference fold R<italic><sup>a</sup></italic>CW. The impact of this substitution resulted in a decrease in the <italic>T</italic><sub>m</sub> by 3°C with an associated ΔΔ<italic>H</italic> = 12 ± 2 kJ·mol<sup>−1</sup> and ΔΔ<italic>G</italic>(0°C) = 1.2 ± 0.5 kJ·mol<sup>−1</sup> (<xref ref-type="table" rid="tbl1">Table 1</xref> and Supplementary Figure S1). Thus, the ‘weak’ CH···N hydrogen bond contributes noticeably to the R-triplex stability.</p><p>The oligonucleotide R<italic><sup>aaa</sup></italic>CW<italic><sup>777</sup></italic> contains three 2AP*(T·7DAA) triplets, and therefore is expected to lose three hydrogen bonds compared to R<italic><sup>a</sup></italic>CW (<xref ref-type="fig" rid="fig1">Figures 1</xref> and <xref ref-type="fig" rid="fig2">2</xref>). Fluorescence is not a method of choice for detection of the denaturation profile of R<italic><sup>aaa</sup></italic>CW<italic><sup>777</sup></italic> since the 2AP fluorescence emission would be affected by energy transfer between the three closely positioned 2-aminopurines. For this reason, the thermal denaturation profile of R<italic><sup>aaa</sup></italic>CW<italic><sup>777</sup></italic> was determined by the UV absorption at 260 nm (Supplementary Figure S2; the derived thermodynamic parameters are presented in <xref ref-type="table" rid="tbl1">Table 1</xref>). As expected, the stability of the R<italic><sup>aaa</sup></italic>CW<italic><sup>777</sup></italic> triplex was exceptionally low, showing a <italic>T</italic><sub>m</sub> 6°C lower than that of R<italic><sup>a</sup></italic>CW with lowered transition enthalpy, entropy and free energy values.</p></sec><sec><title>Additive effect of the hydrogen bonds on the R-triplex stability</title><p>A graphical representation (<xref ref-type="fig" rid="fig5">Figure 5</xref>) of the thermodynamic data (<xref ref-type="table" rid="tbl1">Table 1</xref>) accentuates the energy impact of hydrogen bonds on the R-triplex stability. Both the formation <italic>ΔH</italic>s and <italic>ΔG</italic>s plotted against the predicted number of hydrogen bonds in the R-triplex (relative to the R<italic><sup>a</sup></italic>CW fold) may be fitted with straight lines within the limits of experimental errors. Such a linear dependence implies that the global triplex conformation at low temperature is similar for all of the oligonucleotides; i.e. in the simplest case, the overall stability of the intramolecular DNA fold would be proportional to the number of stabilizing hydrogen bonds, as has been postulated previously for double- and H-form triple-stranded DNA structures (<xref ref-type="bibr" rid="b20">20</xref>,<xref ref-type="bibr" rid="b21">21</xref>).</p><p>One may deduce the contribution of a single H-bond in the R-triplex from these data. A mismatch that removes a single H-bond from the triplex reduces the formation enthalpy by ΔΔ<italic>H</italic> = 18 ± 1.0 kJ·mol<sup>−1</sup> and the formation free energy by ΔΔ<italic>G</italic>(0°C) = 1.8 ± 0.2 kJ·mol<sup>−1</sup> (<xref ref-type="fig" rid="fig5">Figure 5</xref>). As a result, the loss of only three H-bonds in the R<italic><sup>aaa</sup></italic>CW<italic><sup>777</sup></italic> dramatically destabilizes the triplex and leads to a drop in the absolute value of formation enthalpy from 99 ± 3 to 45 ± 3 kJ·mol<sup>−1</sup>(54%) and the free energy from 7.1 ± 0.5 to 2.3 ± 0.7 kJ·mol<sup>−1</sup> (67%). The plots presented in <xref ref-type="fig" rid="fig5">Figure 5</xref> predict that mismatches removing 4–5 hydrogen bonds (two ‘wrong’ bases) from a potential 10 nt long R-triplex would practically abolish triplex formation under our experimental conditions and prevent recognition of the duplex by the third strand. For various DNA duplexes, the absolute values of ΔΔ<italic>G</italic>(0°C) per H-bond estimated from the data given in Ref. (<xref ref-type="bibr" rid="b20">20</xref>) vary from 2 to 10 kJ·mol<sup>−1</sup>. An additional hydrogen bond in the C<sup>+</sup>*(G·C) Hoogsten triplet yielded an additional ΔΔ<italic>G</italic>(25°C) = 5 kJ·mol<sup>−1</sup> (<xref ref-type="bibr" rid="b22">22</xref>,<xref ref-type="bibr" rid="b23">23</xref>). Thus, the R-triplex stability estimated here at ΔΔ<italic>G</italic>(0°C) = 1.8 ± 0.2 kJ·mol<sup>−1</sup> corresponds to the weakest hydrogen bonds observed in nucleic acid base pairs (<xref ref-type="bibr" rid="b24">24</xref>).</p></sec><sec><title>A relatively high contribution of hydrogen bonds to the R-triplex stability</title><p>Unlike the absolute average contribution of H-bonds to the overall DNA structures stability, the relative impact of hydrogen bonds in R-triplex differs significantly from those in duplex and conventional triplex DNA; i.e. the contribution of a single hydrogen bond to the R-triplex formation enthalpy ΔΔ<italic>H</italic>/Δ<italic>H</italic> = 18 kJ·mol<sup>−1</sup>/99 kJ·mol<sup>−1</sup> ≈ 0.17 is quite substantial (<xref ref-type="table" rid="tbl1">Table 1</xref>). The relative free energy contribution of an H-bond to the R-triplex stability is even greater, ΔΔ<italic>G</italic>(0°C)/Δ<italic>G</italic>(0°C) = 1.8 kJ·mol<sup>−1</sup>/7.1 kJ·mol<sup>−1</sup> ≈ 0.25 (<xref ref-type="table" rid="tbl1">Table 1</xref>). In contrast, the relative impact of an additional hydrogen bond to the free energy as well as to formation enthalpy of the H-form triplex for an 11 nt long pyrimidine sequence is 0.09 (<xref ref-type="bibr" rid="b23">23</xref>). These values are close to the corresponding estimations for DNA duplexes (<xref ref-type="bibr" rid="b21">21</xref>,<xref ref-type="bibr" rid="b24">24</xref>,<xref ref-type="bibr" rid="b25">25</xref>).</p><p>The origin of this difference between the R-triplex and the H-triplex is the appreciably lower overall stability of the R-triplex, whose formation enthalpy is only −10 kJ·mol<sup>−1</sup> per base contact (<xref ref-type="table" rid="tbl1">Table 1</xref> and <xref ref-type="fig" rid="fig5">Figure 5</xref>), whereas the average formation enthalpy for the H-triplex (<xref ref-type="bibr" rid="b26">26</xref>–<xref ref-type="bibr" rid="b28">28</xref>) and DNA duplex (<xref ref-type="bibr" rid="b29">29</xref>–<xref ref-type="bibr" rid="b31">31</xref>) are about −30 kJ·mol<sup>−1</sup>. The low stability of the R-triplex is related to its structural features: (i) poor base stacking along the R-strand (<xref ref-type="bibr" rid="b5">5</xref>) and (ii) major ‘sub-grooves’ geometries, that may be less favorable for interactions with ions and water molecules compared with the H-triplex (<xref ref-type="bibr" rid="b32">32</xref>). At the same time, the R-triplex stability is extremely sensitive to the sequence, as stated previously.</p></sec></sec><sec><title>CONCLUSIONS</title><p><list list-type="roman-lower"><list-item><p>The formation of the R-triplex exclusive of an alternative ‘branch migration’ structure has been confirmed directly by strand-specific labeling using the fluorescence base analog 2-aminopurine (2AP).</p></list-item><list-item><p>Strand exchange could be promoted by substitution of two adenines in the third R-strand by 2,6-diaminopurine (DAP) due to a greater stability of the DAP·T base pair in comparison with the A·T base pair.</p></list-item><list-item><p>The hydrogen bonding scheme of the previously proposed (<xref ref-type="bibr" rid="b5">5</xref>) A*(T·A) R-type triplet was strongly supported by the results of adenine substitutions that increased (2,6-diaminopurine) or decreased (7-deazaadenine) the number of hydrogen bonds in the fold.</p></list-item><list-item><p>The relative impact of hydrogen bonds on the overall stability of the R-triplex was significant (compared to the stability of a DNA duplex or an H-triplex).</p></list-item><list-item><p>The large impact of a ‘correct’ base in the third strand opposite the Watson–Crick base pair on the thermodynamic properties of the R-triplex may be a powerful mechanism for ensuring proper recognition of the nucleic acid duplex by the single strand.</p></list-item></list></p></sec><sec><title>SUPPLEMENTARY DATA</title><p>Supplementary Data are available at NAR Online.</p></sec>
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</body><back><ack><p>The authors appreciate helpful discussions with V. L. Florent'ev, M. A. Livshits and V. A. Livshits. The study was supported by RFBR grant 04-04-49618 and the Max Planck Society. 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Biol.</source><year>2000</year><volume>299</volume><fpage>629</fpage><lpage>640</lpage><pub-id pub-id-type="pmid">10835273</pub-id></citation></ref></ref-list><sec sec-type="display-objects"><title>Figures and Tables</title><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Oligonucleotide sequences with the reporting 2AP base, used in the experiments. <italic>a</italic>, 2-aminopurine (2AP); <italic>d</italic>, 2,6-diaminopurine (DAP); <italic>7</italic>, 7-deazaadenine (7DAA). The triplex-forming oligonucleotides: R<italic><sup>a</sup></italic>CW [denoted earlier (<xref ref-type="bibr" rid="b13">13</xref>) as R<sup>2AP</sup>CW] contains 2AP in the third R-strand; RCW<italic><sup>a</sup></italic> contains 2AP in the W-strand; R<italic><sup>add</sup></italic>CW has two adenines in the third R-strand substituted for DAP; R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> has the two adenines in the R-strand and the three adenines in the W-strand substituted for DAP; R<italic><sup>a</sup></italic>CW<italic><sup>7</sup></italic> contains a 2AP*(T·7DAA) triplet with 2AP in the R-strand and 7DAA in place of the adenine in W-strand; R<italic><sup>a</sup></italic>CW<italic><sup>77</sup></italic> contains two A*(T·7DAA) triplets; R<italic><sup>aaa</sup></italic>CW<italic><sup>777</sup></italic> is the triplex with three 2AP*(T·7DAA) triplets. The hairpin-forming oligonucleotide, CW<italic><sup>a</sup></italic>, represents the duplex part of the triplex RCW<italic><sup>a</sup></italic>.</p></caption><graphic xlink:href="gkl431f1"/></fig><fig id="fig2" position="float"><label>Figure 2</label><caption><p>Schematic representation of the isogeometric base triplets accommodating 2-aminopurine (2AP), 2,6-diaminopurine (DAP) and 7-deazaadenine (7DAA).</p></caption><graphic xlink:href="gkl431f2"/></fig><fig id="fig3" position="float"><label>Figure 3</label><caption><p>(<bold>A</bold>) Temperature dependence of the fluorescence emission of 2AP, incorporated in the oligonucleotides: R<italic><sup>a</sup></italic>CW (open circles), RCW<italic><sup>a</sup></italic> (squares), CW<sup>2AP</sup> (diamonds) and R<italic><sup>add</sup></italic>CW (open triangles). Every 10th experimental point is marked. The excitation wavelength was 310 nm, emission was measured at 370 nm. Samples contained 1 µM oligonucleotides, 0.5 M LiCl and 10 mM Tris–HCl buffer, pH 7.6. <italic>Note</italic>: comparison between 2AP in the duplex and in the third strand of the triplex. (<bold>B</bold>) Temperature dependence of the fluorescence emission of 2AP incorporated in the R<italic><sup>a</sup></italic>CW at different concentrations of the oligonucleotide: 1 µM (open circles), 50 µM (filled triangles). The fluorescence was measured in 3 and 1.5 mm cells, respectively.</p></caption><graphic xlink:href="gkl431f3"/></fig><fig id="fig4" position="float"><label>Figure 4</label><caption><p>Temperature dependence of the fluorescence emission of 2AP incorporated in the oligonucleotides: R<italic><sup>a</sup></italic>CW<italic><sup>7</sup></italic> (open triangles), left ordinate; R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> (circles), right ordinate. Solid curves are the best theoretical fits [Materials and Methods and Ref. (<xref ref-type="bibr" rid="b13">13</xref>)]. Derived thermodynamic parameters are given in <xref ref-type="table" rid="tbl1">Table 1</xref>. The experimental conditions are the same as in <xref ref-type="fig" rid="fig3">Figure 3</xref>. Inset: comparison of the R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> melting curves registered with fluorescence (circles, right ordinate) and with absorption at 260 nm (solid curve, left ordinate).</p></caption><graphic xlink:href="gkl431f4"/></fig><fig id="fig5" position="float"><label>Figure 5</label><caption><p>Thermodynamic parameters of the R-triplex formation as functions of the number of predicted hydrogen bonds (compared to R<italic><sup>a</sup></italic>CW). (<bold>A</bold>) The absolute values of the formation enthalpy; (<bold>B</bold>) the absolute values of the formation free energy calculated for 0°C (<xref ref-type="table" rid="tbl1">Table 1</xref>). The triplex folds differ from the R<italic><sup>a</sup></italic>CW either by the number of ‘standard’ NH···N hydrogen bonds (circles), or by the number of the ‘weak’ CH···N bonds (triangles) (for details see <xref ref-type="table" rid="tbl1">Table 1</xref> and main text). Note that all the circles deviate from the straight fitting lines by less than the limits of experimental errors. The only exceptions are the R<italic><sup>a</sup></italic>CW<italic><sup>77</sup></italic> points lying above these lines and having the enthalpy and free energy values relatively close to the R<italic><sup>a</sup></italic>CW values. This is consistent with the CH···N bonds being weaker than the NH···N bonds (<xref ref-type="bibr" rid="b5">5</xref>,<xref ref-type="bibr" rid="b19">19</xref>).</p></caption><graphic xlink:href="gkl431f5"/></fig><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><p>Thermodynamic parameters of the R-triplex formation</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Oligonucleotide</th><th align="left" rowspan="1" colspan="1">Δ<italic>H</italic> (kJ·mol<sup>−1</sup>)</th><th align="left" rowspan="1" colspan="1">Δ<italic>S</italic> (kJ·mol<sup>−1</sup>·deg<sup>−1</sup>)</th><th align="left" rowspan="1" colspan="1"><italic>T</italic><sub>m</sub> (°C)</th><th align="left" rowspan="1" colspan="1">Δ<italic>G</italic>(0°C) (kJ·mol<sup>−1</sup>)</th><th align="left" rowspan="1" colspan="1">Δ(Number of H-bonds) compared with R<italic><sup>a</sup></italic>CW <sup>(a)</sup></th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">R<italic><sup>add</sup></italic>CW<italic><sup>ddd</sup></italic> <sup>(b)</sup></td><td align="left" rowspan="1" colspan="1">−126 ± 7</td><td align="left" rowspan="1" colspan="1">−0.420 ± 0.005</td><td align="left" rowspan="1" colspan="1">27 ± 1</td><td align="left" rowspan="1" colspan="1">−11.3 ± 0.7</td><td align="left" rowspan="1" colspan="1">+2</td></tr><tr><td align="left" rowspan="1" colspan="1">R<italic><sup>a</sup></italic>CW <sup>(b)</sup></td><td align="left" rowspan="1" colspan="1">−99 ± 3</td><td align="left" rowspan="1" colspan="1">−0.337 ± 0.005</td><td align="left" rowspan="1" colspan="1">21 ± 1</td><td align="left" rowspan="1" colspan="1">−7.1 ± 0.5</td><td align="left" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">R<italic><sup>a</sup></italic>CW<italic><sup>7</sup></italic><sup> (b)</sup></td><td align="left" rowspan="1" colspan="1">−84 ± 3</td><td align="left" rowspan="1" colspan="1">−0.289 ± 0.007</td><td align="left" rowspan="1" colspan="1">20 ± 1</td><td align="left" rowspan="1" colspan="1">−5.2 ± 0.7</td><td align="left" rowspan="1" colspan="1">−1</td></tr><tr><td align="left" rowspan="1" colspan="1">R<italic><sup>a</sup></italic>CW<italic><sup>77</sup> </italic><sup>(b,c)</sup></td><td align="left" rowspan="1" colspan="1">−74 ± 2</td><td align="left" rowspan="1" colspan="1">−0.254 ± 0.005</td><td align="left" rowspan="1" colspan="1">18 ± 1</td><td align="left" rowspan="1" colspan="1">−4.6 ± 0.7</td><td align="left" rowspan="1" colspan="1">−2 (‘weak’ H bonds)</td></tr><tr><td align="left" rowspan="1" colspan="1">R<italic><sup>aaa</sup></italic>CW<italic><sup>777</sup></italic> <sup>(d)</sup></td><td align="left" rowspan="1" colspan="1">−45 ± 3</td><td align="left" rowspan="1" colspan="1">−0.153 ± 0.005</td><td align="left" rowspan="1" colspan="1">15 ± 1</td><td align="left" rowspan="1" colspan="1">−2.3 ± 0.7</td><td align="left" rowspan="1" colspan="1">−3</td></tr></tbody></table><table-wrap-foot><fn><p><sup>a</sup>Predicted Δ(Number of H-bonds) between the R-strand and the CW duplex compared with that in R<italic><sup>a</sup></italic>CW, according to the R-triplex model (<xref ref-type="bibr" rid="b5">5</xref>).</p></fn><fn><p><sup>b</sup>Thermodynamic parameters obtained from the fluorescence emission melting curves.</p></fn><fn><p><sup>c</sup>The R<italic><sup>a</sup></italic>CW<italic><sup>77</sup></italic> fold has two A*(T·7DAA) triplets (<xref ref-type="fig" rid="fig2">Figure 2</xref>) which are less stable than the ‘standard’ 2AP*(T·A) triplets, because the former are unable to form ‘weak’ CH···N hydrogen bonds (<xref ref-type="bibr" rid="b5">5</xref>,<xref ref-type="bibr" rid="b19">19</xref>).</p></fn><fn><p><sup>d</sup>Thermodynamic parameters obtained from the UV absorption melting curves.</p></fn></table-wrap-foot></table-wrap></sec></back></article>