Watson-Crick Base-paired Duplex Research Articles

Binding and stabilizations effects of RNA triplex poly(U*A-U) by enantiomers of ruthenium(II) polypyridyl complex [Ru(bpy)2(10-OH-dppz)]2+

Two new chiral ruthenium (II) complexes, Δ-[Ru (bpy)2 (10-OH-dppz)]2+ (Δ-1; bpy = 2,2ʹ-bipyridine, 10-OH-dppz = 10-hydroxyl-dipyrido [3,2-a:2′,3′-c]phenazine) and Λ-[Ru (bpy)2 (10-OH-dppz)]2+ (Λ-1), have been synthesized and characterized in this work. The binding properties of the two enantiomers with an RNA triplex poly (U*A-U) [in text, dash (−) denotes the Watson-Crick base pairing and the asterisk (*) denotes the Hoogsteen base pairing] have been studied. Analysis of the absorption and fluorescence spectra suggest that the binding constant of enantiomer Λ-1 toward the triplex is somewhat smaller than that of enantiomer Δ-1, which is further supported by viscosity experiments. Viscosity experiments showed that although the two enantiomers were intercalated to the triplex, the Δ-enantiomer was a better intercalator than the Λ-enantiomer. Furthermore, thermal denaturation experiments demonstrate that although the two enantiomers can significantly increase the triplex stabilization, unlike enantiomer Λ-1, enantiomer Δ-1 stabilizes the Watson-Crick base-paired duplex and the Hoogsteen base-paired third strand of the triplex with obvious selectivity. In addition, the triplex stabilization by enantiomer Δ-1 was significantly stronger than that by Λ-1. The obtained results indicate that the binding ability of enantiomers Δ-1 and Λ-1 to the triplex is dependent on the chirality of enantiomers.

Binding and stabilization effect of arene ruthenium(Ⅱ) polypyridyl complexes toward the triple-helical RNA poly(U)•poly(A)⁎poly(U)

To determine the binding properties of η6‑arene Ru(Ⅱ) complexes with the RNA triplex poly(U)•poly(A)⁎poly(U), two arene Ru(Ⅱ) complexes, [(ŋ6-C6H6)Ru(m-npip)Cl]+ (Ru1) and [(ŋ6-C6H6)Ru(m-fpip)Cl]+ (Ru2), have been synthesized and characterized in this study. The binding of Ru1 and Ru2 with the triplex RNA poly(U)•poly(A)⁎poly(U) have been investigated by viscosity measurement and various spectroscopic methods. Results demonstrate that the RNA-binding behaviors of Ru1 and Ru2 are intercalation mode. From the analyses of electronic absorption spectra and fluorescence spectra, we observed that the binding affinity for Ru2 is remarkably stronger than that for Ru1. Furthermore, thermal denaturation studies reflect that Ru1 and Ru2 selectively stabilize the RNA triplex, among them, Ru1 can stabilize the Hoogsteen base-paired strand poly(U) (the third strand), while it is little effect on the Watson-Crick base-paired duplex poly(U)•poly(A) (the template duplex). Contrary to Ru1, Ru2 can stabilize both the template duplex and the third-strand, and Ru2 is more effective for stabilization of the third-strand. To our knowledge, this work is the first example of arene metal complexes interacting with triple-helical nucleic acids. The obtained results suggest that subtle differences of substituent on the intercalation ligand may be the important factor affecting on the binding properties of the both arene Ru(Ⅱ) complexes with the RNA triplex, when Ru1 and Ru2 contain the same ancillary ligands.

Chiral ruthenium(II) complexes as stabilizers for an RNA triplex: In contrast to Λ-enantiomer, Δ-enantiomer stabilizes the Watson-Crick duplex and the Hoogsteen strand without significant preference

To further determine the factors governing the triplex stabilization by chiral ruthium(II) polypyridyl complexes, two new dppz-beased binding reagents, Δ-[Ru(bpy)2(6-NO2-dppz)]2+ (Δ-1; bpy = 2,2′-bipyridine; 6-NO2-dppz = 6-nitro-dipyrido-3,2-a,2′,3′-c]phenazine) and Λ-[Ru(bpy)2(6-NO2-dppz)]2+ (Λ-1), have been synthesized and characterized in this work. Binding properties of the two enantiomers Δ-1 and Λ-1 with the RNA poly(U)•poly(A)*poly(U) triplex have been studied by spectroscopic technologies and viscosity measurements as well as melting measurements. Spectral titrations and viscosity experiments as well as light-scattering experiments show that although the two enantiomers bind to the triplex through an intercalative mode, the binding affinity of Δ-1 toward the triplex is slightly higer than that of Λ-1. Moreover, melting measurements show that the two enantiomers exhibit different stabilization effects on the triplex under the same conditions. The enantiomer Δ-1 stabilizes the triplex without significant preference, thereby almost equally stabilizing the Watson-Crick base-paired duplex (the template duplex) and the Hoogsteen base-paired strand (third-strand) of the triplex. In contrast to Δ-1, Λ-1 shows preference for stabilizing the template duplex rather than third-strand to a large extent. This suggests that the racemic complex [Ru(bpy)2(6-NO2-dppz)]2+ is similar as a non-specific metallointercalator for the triplex investigated in this study. The results obtained in this work show that in addition to chiralities of Ru(II) polypyridyl complexes, substituent effects of the main ligands also play important roles in altering the ability and selectivity of the metal complex enantiomers to stabilize the triplex.

Comparative studies on the binding interaction of two chiral Ru(II) polypyridyl complexes with triple- and double-helical forms of RNA

Two chiral Ru(II) polypyridyl complexes, Δ-[Ru(bpy)2(6-F-dppz)]2+ (Δ-1; bpy = 2,2′-bipyridine, 6-F-dppz = 6-fluorodipyrido[3,2-a:2′,3′-c]phenazine) and Λ-[Ru(bpy)2(6-F-dppz)]2+ (Λ-1), have been synthesized and characterized as binders for the RNA poly(U)•poly(A)*poly(U) triplex and poly(A)•poly(U) duplex in this work. Analysis of the UV–Vis absorption spectra and fluorescence emission spectra indicates that the binding of intercalating Δ-1 with the triplex and duplex RNA is greater than that of Λ-1, while the binding affinities of the two enantiomers to triplex structure is stronger than that of duplex structure. Fluorescence titrations show that the two enantiomers can act as molecular “light switches” for triple- and double-helical RNA. Thermal denaturation studies revealed that that the two enantiomers are more stable to Watson-Crick base-paired double strand of the triplex than the Hoogsteen base-paired third strand, but their stability and selectivity are different. For Δ-enantiomer, the increase of the thermal stability of the Watson-Crick base-paired duplex (13 °C) is slightly stronger than of the Hoogsteen base-paired strand (10 °C), displaying no obvious selectivity. However, compared to the Hoogsteen base-paired strand (5 °C), the stability of the Λ-enantiomer to the Watson-Crick base-paired duplex (13 °C) is more significant, which has obvious selectivity. The overall increase in viscosity of the RNA-(Λ-1) system and its curve shape are similar to that of the RNA-(Δ-1) system, suggesting that the binding modes of two enantiomers with RNA are intercalation. The obtained results in this work may be useful for understanding the binding differences in chiral Ru(II) polypyridyl complexes toward RNA triplex and duplex.

A phenolic hydroxyl in the ortho- and meta-positions on the main ligands effect on the interactions of [Ru(phen)2(o-HPIP)]2+ and [Ru(phen)2(m-HPIP)]2+ with the poly(U)·poly(A)*poly(U) triplex

The association of two ruthenium(II) complexes [Ru(phen)2(o-HPIP)]2+ (Ru1; phen = 1,10–phenanthroline, o-HPIP = 2-(2-hydroxyphenyl)-imidazo[4,5-f][1,10] phenanthroline) and [Ru(phen)2(m-HPIP)]2+ (Ru2; m-HPIP = 2-(3-hydroxyphenyl)–imidazo[4,5-f][1,10]phenan-throline) with the RNA poly(U)·poly(A)⁎poly(U) triplex has been investigated by spectrophotometric titrations and melting experiments in this work. All experimental data reveal an intercalative triplex-binding mode of the two complexes, whereas the binding constant for Ru1 is significantly higher than that for Ru2. Circular dichroism spectroscopic investigations show that the two complexes could bind to the chiral environment of the triplex, but the triplex perturbation effects induced by Ru1 are more marked. Thermal denaturation experiments demonstrate that both Ru1 and Ru2 display a large binding preference and stabilizing effect for the third strand over the Watson–Crick base-paired duplex of the triplex. However, the third-strand stabilizing effect of Ru1 is much more effective than that of Ru2. The obtained results suggest that positions of the phenolic group on the main ligands have significant effect on the binding of the two complexes with poly(U)·poly(A)⁎poly(U) triplex

Synthesis and characterization of chiral Ru(II) polypyridyl complexes and their binding and stabilizing effects toward triple-helical RNA

Two novel chiral Ru(II) complexes, Λ- and Δ-[Ru(bpy)2(7-CF3-dppz)]2+ (Λ-1 and Δ-1; bpy = 2,2′-bipyridine, 7-CF3-dppz = 7-trifluoromethyl-dipyrido[3,2-a:2′,3′-c]phenazine), were synthesized and characterized in this work. The binding and stabilizing effects of Λ-1 and Δ-1 toward the RNA poly(U)•poly(A)*poly(U) triplex were studied by various biophysical techniques. Absorption spectra and fluorescence quenching indicates that the binding affinity of Δ-1 is slightly higher than that Λ-1. Both enantiomers induce significant positive viscosity changes that are indicative of intercalative binding, whereas changes in the relative viscosities of the triplex are found to be more pronounced with Δ-1. Melting experiments indicate that the triplex stabilization effects of both enantiomers are significantly different from each other. With Λ-1, the stabilization of the Watson-Crick base-paired duplex (the template duplex) of the triplex shows a moderate increase, whereas the stabilization of the Hoogsteen base-paired strand (third-strand) exhibits slight decrease under the same conditions, suggesting Λ-1 prefers to stabilize the template duplex rather than third-strand. In stark contrast to Λ-1, Δ-1 can not only strongly stabilize the template duplex, but also moderately increase the third-strand stabilization, even so, which imply that Δ-1 also prefer to stabilize the template duplex instead of the third-strand. These suggest that the [Ru(bpy)2(7-CF3-dppz)]2+ is similar as a non-specific metallointercalator the triplex studied in this work. Combined with our recent research, the obtained results further indicate that Δ- enantiomers rather than Λ-ones of Ru(II) polypyridyl complexes usually exhibit stronger binding and stabilizing effects toward the triplex.

Binding properties of two ruthenium(II) polypyridyl complexes [Ru(bpy)2(dppz-Br)]2+ and [Ru(dmb)2(dppz-Br)]2+ with the RNA poly(U)•poly(A)*poly(U) triplex.

Two ruthenium(II) polypyridyl complexes containing different ancillary ligands, [Ru(bpy)2(dppz-Br)]2+ (Ru1; bpy = 2,2'-bipyridine dppz-Br = 7-Br-dipyrido[3,2-a,2',3'-c]-phenazine) and [Ru(dmb)2(dppz-Br)]2+ (Ru2; dmb = 4,4'-dimethyl-2,2'-bipyridine), have been synthesized and characterized. Binding properties of Ru1 and Ru2 with the RNA poly(U)•poly(A)*poly(U) triplex have been investigated by UV-Vis spectroscopy, fluorescence spectroscopy, viscosity measurements as well as circular dichroism and thermal denaturation. Spectrophotometric studies together with viscosity measurements suggest that both Ru1 and Ru2 bind with the triplex by intercalation mode, and the melting experiments demonstrate that the two complexes can effectively enhance the triplex stabilization. However, results indicate that Ru1 stabilizes the third-strand and Watson-Crick base-paired duplex of the triplex without obvious selectivity. In contrast, Ru2 prefers to bind with the third strand rather than the Watson-Crick base-paired duplex of the triplex to a some extent under the same conditions used in this study, thereby significantly stabilizing the third strand. The obtained results of this study suggest that slight differences in the ancillary ligands bpy and dmb should be the main factor affecting the binding interactions of the two complexes with the triplex.

Binding properties of ruthenium(II) complexes [Ru(phen)2(dicnq)]2+ and [Ru(bpy)2(dicnq)]2+ with the RNA triplex poly(U)·poly(A)*poly(U)

Using phen or bpy (phen = 1,10-phenanthroline, bpy = 2,2′-bipyridine) as ancillary ligands and dicnq (dicnq = 6,7-dicyanodipyrido[2,2-d:2′,3′-f]quinoxaline) as the main ligand, two new Ru(II) polypyridyl complexes containing cyano groups, [Ru(phen)2dicnq]2+ (Ru1) and [Ru(bpy)2dicnq]2+ (Ru2) have been synthesized as binders for poly(U)·poly(A)*poly(U) triplex. The binding properties of Ru1 and Ru2 with the triplex has been studied by spectroscopic methods and viscosity measurements. Analysis of the electronic absorption spectra suggests that the binding of intercalating Ru1 with the triplex is greater than that of Ru2, which is also supported by spectroscopic titrations and viscosity measurements. Thermal denaturation studies suggest that the two complexes stabilize the Watson-Crick base-paired duplex of the triplex structure almost without affecting the stability of the Hoogsteen base-paired third strand, suggesting that the electron-withdrawing effect of cyano group hardly increases the binding strengths of the third strand and the template duplex of the triplex under study here. Changes in the CD (CD = circular dichroism) spectra of the triplex indicate that the binding-induced CD perturbation of the triplex structure is more marked with Ru1. The main results obtained here indicate that small differences in the ancillary ligands play important roles in the triplex binding when ruthenium(II) complexes contain the same intercalative ligands.

Ruthenium(II) polypyridyl complex [Ru(phen)2dppz-idzo]2+ as a colorimetric molecular "light switch" and powerful stabilizer for the RNA triplex poly(U)·poly(A)*poly(U).

The interaction of [Ru(phen)2dppz-idzo]2+ (phen = 1,10-phenanthroline, dppz-idzo = dppz-imidazolone) with triplex RNA poly(U)·poly(A)*poly(U) was carried out by using spectroscopic and viscometric techniques in this work. Luminescent titrations suggest that [Ru(phen)2dppz-idzo]2+ shows better selectivity for poly(U)·poly(A)*poly(U) compared with poly(U)·poly(A) and poly(U), this complex exhibits a "light switch" effect with an emission enhancement factor of about 123 in the presence of poly(U)·poly(A)*poly(U). Significantly, this "light switch" behavior could even be observed by the naked eye under irradiation with UV light. To our knowledge, [Ru(bpy)2dppz-idzo]2+ is the first small molecule able to serve as a colorimetric molecular "light switch" for the triplex poly(U)·poly(A)*poly(U). Combined with the spectral and viscometric results as well as [Ru(phen)2dppz-idzo]2+ stabilizing the template duplex poly(U)·poly(A), we speculate that [Ru(phen)2dppz-idzo]2+ prefers to bind with the Hoogsteen base-paired strand (the third strand) of the triplex, thus the intercalating [Ru(phen)2dppz-idzo]2+ stabilizing the third strand is more marked in comparison with the Watson-Crick base-paired duplex of the triplex. The results obtained here may be useful for understanding the interaction of triplex RNA poly(U)·poly(A)*poly(U) with small molecule, particularly ruthenium(II) complexes.

Ru(bpy)2(7-CH3-dppz)](2+) and [Ru(phen)2(7-CH3-dppz)](2+) as metallointercalators that affect third-strand stabilization of the poly(U)˙poly(A)*poly(U) triplex.

Stable RNA triplexes play key roles in many biological processes. However, due to Hoogsteen base pairing, triplexes are thermodynamically less stable than the corresponding duplexes. To understand the factors effecting the stabilization of RNA triplexes by octahedral ruthenium(ii) complexes, two Ru(ii) complexes, [Ru(bpy)2(7-CH3-dppz)](2+) (Ru) and [Ru(phen)2(7-CH3-dppz)](2+) (Ru), have been synthesized and characterized in this work. The interactions of the two Ru(ii) complexes with the poly(U)˙poly(A)*poly(U) triplex are investigated by spectrophotometry, spectrofluorometry, circular dichroism as well as viscometry. The results demonstrate that the two complexes are able to enhance the stability of the RNA triplex and serve as molecular "light switches" for the triplex. However, Ru and Ru affecting the stabilization of the third strand are significantly weaker than that of the Watson-Crick base-paired duplex, suggesting that the binding of the two complexes with the triplex is favored by the Watson-Crick base-paired duplex to a large extent. In addition, considering the nature of Ru and Ru, we presume that their binding differences may be due to different ancillary ligand effects. This study further advances our knowledge on the interaction of RNA triple-stranded structures with metal complexes, particularly with Ru(ii) complexes.

ESI-MS Investigation of an Equilibrium between a Bimolecular Quadruplex DNA and a Duplex DNA/RNA Hybrid.

Electrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the quadruplex DNA (qDNA) resulted in formation of the duplex DNA/RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine (1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its circular dichroism (CD) melting temperature of 10°C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of (1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid. Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if telomeric DNA was folded into a qDNA structure, it is possible for a DNA/RNA hybrid to form as is required during template alignment. The discrimination of ligand (1) for binding to the bimolecular qDNA over the DNA/RNA hybrid positions it as a useful compound for probing the role(s), if any, of antiparallel qDNA in the telomerase mechanism.

Stabilization of a Bimolecular Triplex by 3'-S-Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation.

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single-stranded nucleic acids are also relevant in third-strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson-Crick (W-C) base-paired DNA duplexes and a Hoogsteen base-paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA-like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3'-S-phosphorothiolate (3'-SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. (1) H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3'-SP modifications is more compact. The 3'-SP modification was previously shown to stabilise G-quadruplex and i-motif structures and therefore is now proposed as a generic solution to stabilising multi-stranded DNA structures.

Effect of ancillary ligands on the interaction of ruthenium(II) complexes with the triplex RNA poly(U)·poly(A)*poly(U)

Two new Ru(II) complexes with 1,8-naphthalimide group, [Ru(phen)2(pnip)]2+ (Ru1; phen=1,10-phenanthroline, pnip=2-[N-(p-phenyl)-1,8-napthalimide]imidazo[4',5'-f][1,10]phenanthroline) and [Ru(bpy)2(pnip)]2+ (Ru2; bpy=2,2′-bipyridine), have been synthesized and characterized. The interactions of Ru1 and Ru2 with the triplex RNA poly(U)•poly(A)*poly(U) (where • denotes the Watson–Crick base pairing and * denotes the Hoogsteen base pairing) were studied by various biophysical. Electronic spectra established that the binding affinity for Ru1 was greater than that for Ru2. Fluorescence and viscosity studies gave convincing evidence for a true intercalative binding of both complexes with the RNA triplex. UV melting studies confirmed that the two complexes could stabilize the triplex, whereas the effects of the two complexes on the stability of the Hoogsteen base-paired strand ploy(U) and the Watson–Crick base-paired duplex poly(U)•poly(A) of the triplex were different. In the case of Ru1, the increase of the thermal stability of the Hoogsteen base-paired strand was stronger than that of the Watson–Crick base-paired duplex. However, an opposite effect was observed in the case of Ru2. Circular dichroic studies suggested that the RNA triplex undergoes a conformational transition in the presence of Ru1, whereas the helicity of the RNA triplex still remains A-type in the presence of Ru2. The main results obtained here further advance our knowledge on the interaction of RNA triple-stranded structures with metal complexes, particularly ruthenium(II) complexes.

Charge Conduction Properties of a Parallel-Stranded DNA G-Quadruplex: Implications for Chromosomal Oxidative Damage

The charge-flow properties and concomitant guanine damage patterns of a number of intermolecular and wholly parallel-stranded DNA G-quadruplexes were investigated. The DNA constructs were structurally well-defined and consisted of the G-quadruplex sandwiched and stacked between two Watson-Crick base-paired duplexes. Such duplex-quadruplex-duplex constructs were designed to minimize torsional stress as well as steric crowding at the duplex-quadruplex junctions. When anthraquinone was used to induce charge flow within the constructs, it was found that the quadruplex served both as a sink and as a moderately good conductor of electron holes, relative to DNA duplexes. Most strikingly, the quadruplex suffered very little charge-flow generated oxidative damage relative to guanines in the duplex regions and, indeed, to guanines in antiparallel quadruplexes reported in prior studies. It is likely that these differences result from a combination of steric and electronic factors. A biological conclusion that may be drawn from these data is that if, as anticipated, G-quadruplex structures form in vivo at the telomeres and other loci in eukaryotic chromosomes, their ability to serve as protective sinks against chromosomal oxidative damage may depend on their specific character and topology. From a separate perspective, our results on the conduction properties of duplex-quadruplex-duplex DNA composites suggest the utility of G-quadruplexes as junction modules in the construction of DNA-based biosensors and nanocircuitry.

The AUUCU Repeats Responsible for Spinocerebellar Ataxia Type 10 Form Unusual RNA Hairpins

Spinocerebellar ataxia type 10 is an autosomal dominant disorder caused by expansion of a pentameric repeat tract (ATTCT.AGAAT)(n) in intron 9 of the gene that encodes ataxin-10. We have analyzed duplex DNA containing the repeat, the individual DNA strands, and the RNA that would be generated by transcription of the repeat. Circular dichroism and UV absorbance measurements suggest that the previously reported tendency of the repeat to unpair when supercoiled is probably related simply to GC content rather than reflecting any unusual property of the duplex. DNA containing d(ATTCT)9 forms a folded structure at relatively low temperatures, whereas the antisense strand, d(AGAAT)9, does not form a structure even at 0 degrees C. In contrast r(AUUCU)9 forms a folded structure under physiologically reasonable conditions. S1 nuclease analysis reveals a single region of hypersensitivity in the middle of the repeat tract, whereas V1 digestion is consistent with a hydrogen bonded or well stacked structure. CD spectroscopy shows that the structure is unimolecular and hydrogen bonded and has a significant amount of A-form helix. NMR spectroscopy demonstrates that these hydrogen bonds comprise an equal number of A.U and U.U base pairs. Our data thus suggest that the repeat forms an unusual RNA hairpin. Thus the ability to form an RNA hairpin seems to be a common property of those Repeat Expansion Diseases that are not recessively inherited and are caused by repeats that are transcribed but not translated.

The First Example of a Hoogsteen Basepaired DNA Duplex in Dynamic Equilibrium with a Watson-Crick Basepaired Duplex—A Structural (NMR), Kinetic and Thermodynamic Study

A single-point substitution of the O4′ oxygen by a CH2 group at the sugar residue of A 6 (i.e. 2′-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5′- d(C1G2C3G4A5A6T7T8C9G10C11G12)2 −3, has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A 6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)→←(1B): Keq = k1/k-1 = 0.56±0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9± 0.8 sec−1; δH°‡ = 164±14 kJ/mol;-TδS°‡ (298K) = −92 kJ/mol giving a δG298°‡ of 72 kJ/mol. Ea (k1) = 167±14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0±0.6 sec-1, δH°‡ = 153±13 kJ/mol;-TδS°‡ (298K) = −82 kJ/mol giving a δG298°‡ of 71 kJ/mol. Ea (k-1) = 155±13 kJ/mol]. A comparison of δG298°‡ of the forward (k1) and backward (k-1) conversions, (1A)→←(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A 6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A 6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2′- deoxyaristeromycin moiety, A 6, as a result of substitution of the endocyclic 4′-oxygen in the natural sugar with a methylene group in A 6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in α, σ and γ torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.

The crystal structure of the octamer [r(guauaca)dC]2 with six Watson-Crick base-pairs and two 3′ overhang residues

The crystal structure of an alternating RNA octamer, r(guauaca)dC (RNA bases are in lower case while the only DNA base is in upper case), with two 3′ overhang residues one of them a terminal deoxycytosine and the other a ribose adenine, has been determined at 2.2 Å resolution. The refined structure has an Rwork 18.6 % and Rfree 26.8 %. There are two independent duplexes (molecules I and II) in the asymmetric unit cell, a = 24.95, b = 45.25 and c = 73.67 Å, with space group P212121. Instead of forming a blunt end duplex with two a+·c mispairs and six Watson-Crick base-pairs, the strands in the duplex slide towards the 3′ direction forming a two-base overhang (radC) and a six Watson-Crick base-paired duplex. The duplexes are bent (molecule I, 20°; molecule II, 25°) and stack head-to-head to form a right-handed superhelix. The overhang residues are looped out and the penultimate adenines of the two residues at the top end (A15) are anti and at the bottom (A7) end are syn. The syn adenine bases form minor groove A∗(G·C) base triples with C8-H···N2 hydrogen bonds. The anti adenine in molecule II also forms a triple and a different C2-H···N3 hydrogen bond, while the other anti adenine in molecule I does not, it stacks on the looped out overhang base dC. The 3′ terminal deoxycytosines form two stacked hemiprotonated trans d(C·C)+ base-pairs and the pseudo dyad related molecules form four consecutive deoxyribose and ribose zipper hydrogen bonds in the minor groove.

Solution structure of the esperamicin A 1-DNA complex

Esperamicin A 1 is an enediyne antibiotic possessing antitumor activity associated with its ability to bind and, following activation, affect strand cleavage of DNA. We report on the solution structure of the esperamicin A 1-d(C-G-G-A-T-C-C-G) duplex complex based on a combined analysis of NMR and molecular dynamics calculations including intensity refinement in a water box. The refined solution structures of the complex provide a molecular explanation of the sequence specificity for binding and cleavage by this member of the enediyne family of antitumor antibiotics. Esperamycin A 1 binds to the DNA minor groove with its methoxyacrylyl- anthranilate moiety intercalating into the helix at the (G2-G3)·(C6′-C7′) step. The methoxyacrylyl-anthranilate intercalator and the minor groove binding A-B-C trisaccharide moieties rigidly anchor the enediyne in the minor groove such that the pro-radical centers of the enediyne are proximal to their anticipated proton abstraction sites. Specifically, the pro-radical C-3 and C-6 atoms are aligned opposite the abstractable H-5′ (pro-S) proton of C6 and the H-1′ proton of C6′ on partner strands, respectively, in the complex. The thiomethyl sugar B residue is buried deep in an edgewise manner in the minor groove with its two faces sandwiched between the walls of the groove. Further, the polarizable sulfur atom of the thiomethyl group of sugar B residue is positioned opposite and can hydrogen-bond to the exposed amino proton of G3′ in the complex. There is little perturbation away from a right-handed Watson-Crick base-paired duplex in the complex other than unwinding of the helix at the intercalation site and widening of the minor groove centered about the enediyne-binding and anthranilate intercalation sites. Sequence-specific binding of esperamycin A 1 to the d(C-G-G-A-T-C-C-G) duplex is favored by the complementarity of the fit between the drug and the floor of the minor groove, good stacking between the intercalating anthranilate ring and flanking purine bases and intermolecular hydrogen-bonding interactions.

Induced Chirality in PNA-PNA Duplexes

Complementary peptide nucleic acids (PNA) form Watson-Crick base-paired helical duplexes. The preferred helicity of such a duplex is determined by a chiral amino acid attached to the C-terminus. We here show that the induced helicity, as measured by circular dichroism (CD), is drastically dependent on the nucleobase sequence proximal to the chiral center. Chemically linked PNA tetramer duplexes of all 16 combinations of the two bases proximal to a carboxy terminal lysine residue were studied by CD. We conclude that the base next to the chiral center must be either a guanine or a cytosine for efficient stabilization of one helical sense. In case of cytosine, the subsequent base should preferably be a purine. We also show that the side chain properties of the C-terminal amino acid influence the resulting sense of helicity. The propagation length of induced chirality in PNA duplexes is found to be around 10 base pairs. Theoretical calculations of the circular dichroism for B-DNA, using the quantum mechanical matrix method of Schellman, give spectra in reasonable agreement with those found experimentally for PNA duplexes. The rate of helix conversion of the duplexes shows first-order kinetics with a rate constant in the range of minutes. Shorter duplexes are found to have lower activation energy and larger negative activation entropy for helix conversion, in agreement with a conversion mechanism in which a perfect helix is switched to the opposite handedness.

Peptide nucleic acid (PNA) conformation and polymorphism in PNA-DNA and PNA-RNA hybrids.

Two hydrogen-bonding motifs have been proposed to account for the extraordinary stability of polyamide "peptide" nucleic acid (PNA) hybrids with nucleic acids. These interresidue- and intraresidue-hydrogen-bond motifs were investigated by molecular mechanics calculations. Energy-minimized structures of Watson-Crick base-paired decameric duplexes of PNA with A-, B-, and Z-DNA and A-RNA polymorphs indicate that the inherent stability of the complementary PNA helical structures is derived from interresidue, rather than from intraresidue, hydrogen bonds in all hybrids studied. Intraresidue-hydrogen-bond lengths are consistently longer than interresidue hydrogen bonds. Helical strand stability with interresidue hydrogen bond stabilization follows the order: B-(DNA.PNA) > A-(DNA.PNA) congruent to A-RNA.PNA > Z-(DNA.PNA). In the triplex hybrids A-(RNA.PNA2) and B-(DNA.PNA2), differences between stabilities of the two decamers of thyminyl PNA with lysine amide attached to the C terminus (pnaT)10 strands are small. The Hoogsteen (pnaT)10 strands are of slightly higher potential energy than are the Watson-Crick (pnaT)10 strands. Antiparallel arrangement of PNAs in the triplex is slightly favored over the parallel arrangement based on the calculations. Examination by molecular mechanics of the PNA.DNA analogue of the NMR-derived structure for the B-double-stranded DNA dodecamer d(CG-CAAATTTGCG)2 in solution suggests that use of all bases of the genetic alphabet should be possible without loss of the specific interresidue-hydrogen-bonding pattern within the PNA strand.