NMR Study of Complexation of Aromatic Ligands with Heptadeoxynucleotide 5"-d(GCGAAGC) Forming Stable Hairpin Structure in Aqueous Solution

Complex formation of hairpin-producing heptadeoxynucleotide 5"-d(GCGAAGC) with aromatic molecules: acridine dye proflavine and anthracycline antibiotic daunomycin was studied by one-dimensional ¹H NMR and two-dimensional correlation ¹H–¹H (2D-TOCSY, 2D-NOESY), ¹H–³¹P (2D-HMBC) NMR spectroscopy (500 and 600 MHz) in aqueous solution. Concentration and temperature dependences for the chemical shifts of ligand protons were measured, molecular models of equilibrium in solution were developed, and equilibrium thermodynamic parameters for the formation of intercalation complexes were calculated. Spatial structures of dye and antibiotic complexes with the heptamer hairpin were constructed on the basis of 2D-NOE data and the calculated values of limiting chemical shifts of ligand protons.     

NMR study of complex formation of aromatic ligands with heptadeoxynucleotide 5'-d(GCGAAGC) forming stable hairpin structure in aqueous solution

Complex formation of hairpin-producing heptadeoxynucleotide 5'-d(GCGAAGC) with aromatic molecules: acridine dye proflavine and anthracycline antibiotic daunomycin was studied by one-dimensional 1H NMR and two-dimensional correlation 1H-1H (2M-TOCSY, 2M-NOESY), 1H-31P (2M-HMBC) NMR spectroscopy (500 and 600 MHz) in aqueous solution. Concentration and temperature dependences for the chemical shifts of ligand protons were measured, molecular models of equilibrium in solution were developed, and equilibrium thermodynamic parameters for the formation of intercalation complexes were calculated. Spatial structures of dye and antibiotic complexes with the heptamer hairpin were constructed on the basis of 2M-NOE data and the calculated values of limiting chemical shifts of ligand protons.     

Study of the complex formation of daunomycin with deoxytetranucleotides with bases of differing sequence in an aqueous solution by 1H-NMR spectroscopy

The complex formation of the antibiotic daunomycin with deoxytetranucleotides of different base sequence in the chain, 5'-d(GpCpGpC), 5'-d(CpGpCpG), and 5'-d(TpGpCpA) in aqueous salt solution was studied by 1D and 2D (2M-TOCSY and 2M-NOESY) 1H-NMR spectroscopy. Concentration and temperature dependences of proton chemical shifts of molecules were measured. Based on these dependences, reaction equilibrium constants, relative content of various complexes depending on concentration and temperature, limiting values of chemical shifts of protons of daunomycin incorporated in various complexes, and the thermodynamic parameters delta H and delta S of complex formation were calculated. The analysis of the results enables the conclusion that the sites of predominant intercalation of daunomycin are triplet nucleotide sequences, the binding sites of the antibiotic with three consecutive GC pairs in the tetranucleotide duplex being more preferential. Daunomycin exhibits no sequence specificity upon binding to the single-stranded deoxynucleotide sequence. From the calculated values of induced chemical shifts of daunomycin protons and 2M-NOE data, the most probable spatial structures of complexes (1:2) of the antibiotic with deoxytetranucleotides were constructed. The binding of the second daunomycin molecule to both the single-stranded and duplex form of tetramers is of pronounced anticooperative mode, which is explained by the presence in the antibiotic of a positively charged amino sugar residue, which poses considerable steric constraints for the insertion of the second antibiotic molecule into the short tetranucleotide sequence. The results were compared with the data obtained under identical experimental conditions for typical intercalators proflavine and ethidium bromide.     

1H-NMR structural analysis of ethidium bromide complexation with self-complementary deoxytetranucleotides 5′-d (ApCpGpT), 5′-d (ApGpCpT), and 5′-d (TpGpCpA) in aqueous solution

Complexation of the trypanocidal drug, ethidium bromide (EB), and the self-complementary deoxytetraribonucleoside triphosphates, 5′-d(ApCpGpT), 5′-d(ApGpCpT), and 5′-d(TpGpCpA), in aqueous salt solution has been investigated using one-dimensional and two-dimensional 500/600 MHz ¹H-nmr spectroscopy. Six hundred megahertz two-dimensional homonuclear ¹H-nmr spectroscopy (nuclear Overhauser effect spectroscopy) was used for a qualitative determination of the structures of EB binding with the deoxytetranucleotides. Concentration dependencies of proton chemical shifts of the molecules have been measured at constant temperatures (T = 303 or 308 K). Different successive schemes of complex formation between the dye molecule and the tetranucleotides have been examined by taking into account various molecular associations in solution, viz., 1:1, 1:2, 2:1 and 2:2 complexes. Equilibrium reaction constants and the limiting proton chemical shifts in the complexes have been determined. The relative contributions of different types of complexes in the equilibrium mixture have been determined and special features of the dynamic equilibrium have been revealed by analysis of chemical shifts as a function of both the dye and tetranucleotide concentrations. The present analysis leads to the conclusion that EB binds preferentially to the pyrimidine-purine sites of the tetranucleotide duplexes. The results show that the energy of EB binding depends on the base content in the pyrimidine-purine sites of the tetramers and on the nucleotide residuals flanking the preferential site. The most favorable structures of the 1:2 and 2:2 complexes of the dye with the tetranucleotides have been constructed using calculated values of induced chemical shifts of EB protons in conjunction with intermolecular nuclear Overhauser effects. The structures of the EB:tetranucleotide complexes depend on tetramer base sequence and are characterized by differences in helix parameters. © 1996 John Wiley & Sons, Inc.     

Thermodynamic analysis of interaction of mitoxantrone with deoxytetranucleotide 5'-d(TpGpCpA) in the water solution based on 1H-NMR spectrophotometry

The complex formation of the antibiotic mitoxantrone (novantrone) with the deoxytetranucleotide 5'-d(TpGpCpA) in an aqueous salt solution was studied by one- and two-dimensional (2D-TOSCY and 2D-NOESY) 1H NMR spectroscopy (500 MHz). Concentration and temperature dependence of proton chemical shifts of molecules were measured. On the basis of these data, the equilibrium constants of the reaction, the relative content of various complexes as a function of concentration and temperature, the limiting values of chemical shifts of novantrone in complexes, and the thermodynamic parameters delta H and delta S of complex formation of molecules were calculated. It was concluded that the attachment sites for novantrone are pyrimidine-purine nucleotide sequences, sites d(TG) and d(CA) of the tetranucleotide duplex. The analysis of the thermodynamic parameters of the complex formation suggests that intermolecular hydrogen bonds and electrostatic interactions of the aminoalkyl chains of novantrone with the duplex d(TpGpCpA)2 play an important role in the stabilization of complexes 1:2 and 2:2. The results were compared with those obtained earlier for typical intercalators of ethidium bromide and daunomycin under identical experimental conditions.     

Thermodynamic analysis of the interaction of the acridine dye proflavin with deoxytetranucleotides with various base sequences from (1)H-NMR data]

Temperature dependences of proton chemical shifts of the molecules in aqueous solutions of proflavine with deoxytetraribonucleoside triphosphates of different base sequence--5'-d(CGCG), 5'-d(GCGC), 5'-d(ACGT) and 5'-d(AGCT) have been studied by 500 MHz NMR spectroscopy. Methods of calculation of thermodynamical parameters of complex formation, giving an opportunity to differentiate the contributions of the reactions leading to the formation of different types of complexes in conditions of the composite equilibrium in solution, have been suggested. Enthalpies and entropies of the reactions of 1:1, 2:1, 1:2, 2:2 complexes formation between proflavine and tetranucleotides have been determined. Comparative analysis of the calculated thermodynamical parameters has been made and assumptions about the nature of intermolecular interactions responsible for the formation of dye complexes with different tetranucleotides have been proposed.     

1H NMR Thermodynamical Analysis of the Interactions of Proflavine with Self-Complementary Deoxytetranucleotides of Different Base Sequence

Abstract

Enthalpies and entropies of complex formation (1:1, 2:1, 1:2, and 2:2 complexes) between proflavine and tetranucleotides of different base sequence have been determined by 500 MHz proton NMR chemical shifts, enabling the contributions to be differentiated for the formation of different types of complexes in solution. Comparison of the calculated thermodynamical parameters has lead to an understanding of the nature of the intermolecular interactions responsible for the formation of dye complexes with the different tetranucleotides.     

Studies by means of 1H NMR spectroscopy of complex formation of aromatic biologically active compounds with antibiotic topotecan

The analysis of heteroassociation of antibiotic topotecan (TPT) with aromatic biologically active compounds (BAC): caffeine, mutagens ethidium bromide and proflavine, antibiotic daunomycin, vitamins flavin-mononucleotide and nicotinamide, has been carried out in the work using 1H NMR spectroscopy data. The equilibrium constants of heteroassociation and induced chemical shifts of the protons have been obtained in the complexes with BAC. It is found that the complex formation TPT-BAC has the nature of stacking of the chromophores, additionally stabilized in the case of proflavine by intermolecular hydrogen bond. Calculation of the basic components of the Gibbs free energy of the complexation reactions is carried out, and the factors which stabilize and destabilize the heterocomplexes of molecules are revealed.     

NMR studies of echinomycin bisintercalation complexes with d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution: sequence-dependent formation of Hoogsteen A1.T4 and Watson--Crick T1.A4 base pairs flanking the bisintercalation site

We report on two-dimensional proton NMR studies of echinomycin complexes with the self-complementary d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution. The exchangeable and nonexchangeable antibiotic and nucleic acid protons in the 1 echinomycin per tetranucleotide duplex complexes have been assigned from analyses of scalar coupling and distance connectivities in two-dimensional data sets recorded in H2O and D2O solution. An analysis of the intermolecular NOE patterns for both complexes combined with large upfield imino proton and large downfield phosphorus complexation chemical shift changes demonstrates that the two quinoxaline chromophores of echinomycin bisintercalate into the minor groove surrounding the dC-dG step of each tetranucleotide duplex. Further, the quinoxaline rings selectively stack between A1 and C2 bases in the d(ACGT) complex and between T1 and C2 bases in the d(TCGA) complex. The intermolecular NOE patterns and the base and sugar proton chemical shifts for residues C2 and G3 are virtually identical for the d(ACGT) and d(TCGA) complexes. A change in sugar pucker from the C2'-endo range to the C3'-endo range is detected at C2 on formation of the d(ACGT) and d(TCGA) complexes. In addition, the sugar ring protons of C2 exhibit upfield shifts and a large 1 ppm separation between the H2' and H2" protons for both complexes. The L-Ala amide protons undergo large downfield complexation shifts consistent with their participation in intermolecular hydrogen bonds for both tetranucleotide complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H-NMR analysis of heteroassociation of caffeine with antibiotic actinomycin D in the aqueous solution

The molecular basis of the action of caffeine as a complex forming agent, an interceptor of aromatic drugs intercalating into DNA was studied by the example of the an anticancer antibiotic actinomycin D examined. The hetero-association of caffeine and actionomycin D was studied by one- and two-dimensional 1H-NMR spectroscopy (500 MHz). Concentration and temperature dependences of the proton chemical shifts of molecules in aqueous solution were measured. The equilibrium reaction constant of hetero-association of caffeine with actinomycin D (K = 246 +/- 48 M-1), the limiting chemical shifts of caffeine protons in complexes were determined. The most favourable structure of the 1:1 caffeine-actinomycin D hetero-complex in aqueous solution was constructed using the calculated values of the induced proton chemical shifts of molecules and the quantum-mechanical iso-shielding curves for caffeine and actinomycin D. The thermo-dynamical parameters of the hetero-complex formation between caffeine and actinomycin D were also determined. The structural and thermo-dynamical analysis showed that dispersive forces and hydrophobic interactions play the major role in hetero-association of caffeine and actinomycin D in aqueous-salt solution. The relative content of different complexes in mixed solutions containing caffeine and actinomycin D was calculated and distinctive features of the dynamic equilibrium of caffeine-actinomycin D hetero-associates were revealed as a function of concentration and temperature. It is concluded that hetero-association of caffeine and actinomycin D molecules a lowers the effective concentration of the drug in solution and hence the pharmacological activity of actinomycin D.     

Study of the interaction of proflavine and isomeric diribonucleoside monophosphates CpG and GpC by proton magnetic resonance

A comparative study of the interaction of proflavine with isomeric diribonucleoside monophosphates CpG and GpC has been made by the method of 1H NMR (270 MHz). A method of calculation of the parameters of complex formation from the concentration dependences of proton chemical shifts of the dye has been proposed. The equilibrium constants of 1:1 and 1:2 complexes association of these molecules and the most probable structures of the complexes have been determined.     

Determination of thermodynamic parameters of proflavine interaction with ribodinucleoside monophosphates CpG and PpC in an aqueous solution from data of proton magnetic resonance

The temperature dependence of proton chemical shifts of proflavine in solution with isomeric ribodinucleoside monophosphates CpG and GpC has been studied. A scheme of calculation of thermodynamic parameters of dye aggregation and 1 : 1 and 1 : 2 complexes formation with dinucleotides has been proposed. The Gibbs' free energy, enthalpy and entropy of these reactions have been determined. It is concluded that hydrophobic interactions play an important part when 1 : 2 complex of proflavine with ribonucleosides is being formed.     

Interaction of proflavin with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT): thermodynamic analysis from (1)H NMR data]

Temperature relationships of chemical shifts of protons of proflavin mixed with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in water solution were investigated on impulse NMR spectrometer (500 MHz). Procedure is suggested for calculating values of mole fractions of various associates in solution as a function against temperature. Free energies of Gibbs, entalpy and entropy were determined in the reactions of complex formation 1:1, 1:2, 2:1 of proflavin with tetranucleotide. The results point to a significant role of hydrophobic interactions during the formation of dye--tetramere duplex complexes.     

Mutagen–nucleic acid complexes at the polynucleotide duplex level in solution: Intercalation of proflavine into poly(dA-dT) and the melting transition of the complex

The nmr chemical shifts and line widths of the nucleic acid base and sugar proton resonances and the proflavine ring protons can be monitored through the melting transition of the proflavine + poly(dA-dT) complex, phosphate/dye (P/D) ratio = 24 and 8 in 1M salt solution. The nucleic acid and mutagen protons in the complex are in fast exchange between duplex and strand states with the midpoint of the melting transition monitored at the nucleic acid resonances increasing from 72.6°C for poly(dA-dT) to 78.1°C for the P/D = 24 complex and 83.4°C for the P/D = 8 complex in 1M salt solution. The melting transition monitored by the proflavine resonances were 80.0°C for the P/D = 24 complex and 84.3°C for the P/D = 8 complex in 1M salt solution. Since the nucleic acid is in excess at high P/D ratios, the nucleic acid transitions are an average for the opening of mutagen-free and mutagen-bound base-pair regions, while the proflavine transitions monitor the melting of mutagen-bound base-pair regions. The observed 0.75 to 0.95 ppm unfield shift at all four proflavine protons on formation of the complex with poly(dA-dT) provides direct evidence for intercalation of the mutagen between base pairs of the nucleic acid duplex. We have deduced the approximate overlap geometry between the proflavine ring and nearest-neighbor base pairs at the intercalation site from a comparison between experimental proflavine complexation shifts and those calculated for various stacking orientations. The experimental chemical shift of the poly(dA-dT) adenine H-2 resonance in the duplex state in the absence and presence of proflavine suggests that intercalation occurs preferentially at dT-dA sites. The selective chemical shift changes at the sugar H-2′,2″ and H-3′ resonances of the poly(dA-dT) duplex on complex formation demonstrates changes in the sugar pucker and/or torsion angles of the sugar phosphate backbone at the intercalation site.     

NMR investigation of the complexation of daunomycin with deoxytetranucleotides of different base sequence in aqueous solution

500 MHz NMR spectroscopy has been used to investigate the complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary deoxytetranucleotides, 5'-d(CGCG), 5'-d(GCGC), 5'-d(TGCA), 5'-d(ACGT) and 5'-d(AGCT), of different base sequence in aqueous salt solution. 2D homonuclear 1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H - 31P NMR spectroscopy (HMBC) have been used for complete assignment of the non-exchangeable protons and the phosphorus resonance signals, respectively, and for a qualitative determination of the preferred binding sites of the drug. Analysis shows that DAU intercalates preferentially into the terminal sites of each of the tetranucleotides and that the aminosugar of the antibiotic is situated in the minor groove of the tetramer duplex, partly eclipsing the third base pair. A quantitative determination of the complexation of DAU with the deoxytetranucleotides has been made using the experimental concentration and temperature dependences of the drug proton chemical shifts; these have been analysed in terms of the equilibrium reaction constants, limiting proton chemical shifts and thermodynamical parameters (enthalpies deltaH, entropies deltaS) of different drug-DNA complexes (1:1, 1:2, 2:1, 2:2) in aqueous solution. It is found that DAU interacts with sites containing three adjacent base pairs but does not show any significant sequence specificity of binding with either single or double-stranded tetranucleotides, in contrast with other intercalating drugs such as proflavine, ethidium bromide and actinomycin D. The most favourable structures of the 1:2 complexes have been derived from the induced limiting proton chemical shifts of the drug in the intercalated complexes with the tetranucleotide duplex, in conjunction with 2D NOE data. It has been found that the conformational parameters of the double helix and the orientation of the DAU chromophore in the intercalated complexes depend on base sequence at the binding site of the tetramer duplexes in aqueous solution.     

Hetero-association of caffeine and aromatic drugs and their competitive binding with a DNA oligomer

NMR spectroscopy has been used to elucidate the molecular basis of the action of caffeine (CAF) on the complexation with DNA of mutagens such as ethidium bromide, propidium iodide, proflavine and acridine orange, and anticancer drugs such as actinomycin D and daunomycin. The hetero-association of CAF and each of the aromatic ligands in 0.1 mol L(-1) phosphate buffer (pD=7.1) has been investigated as a function of concentration and temperature by 500 MHz 1H NMR spectroscopy and analysed in terms of a statistical-thermodynamic model, in which molecules form indefinite aggregates for both self-association and hetero-association. The analysis leads to determination of the equilibrium constants of hetero-association and to the values of the limiting chemical shifts of the heteroassociation of CAF with each of the aromatic molecules. The hetero-association constants between CAF and each of the aromatic drugs/dyes are found to be intermediate in magnitude between those for self-association of CAF and the corresponding drug/dye. The most probable structures of the 1:1 CAF + ligand hetero-association complexes have been determined from the calculated values of the induced limiting chemical shifts of the drug protons. Knowledge of the equilibrium constants for self-association of CAF and the aromatic ligands, for their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the CAF-ligand and CAF-ligand-d(TGCA) complexes to be calculated as a function of CAF concentration in mixed solutions. It is concluded that, on addition of CAF to the solution, the decrease in binding of drug or mutagen with DNA is due both to competition for the binding sites by CAF and the aromatic molecules, and to formation of CAF-ligand hetero-association complexes in the mixed solution; the relative importance of each process depends on the drug or mutagen being considered.     

Distinctive features of the self-association of deoxyhexanucleotide 5'-D(GpCpApTpGpC) in aqueous solution: 1H NMR analysis

The self-association of self-complementary deoxyhexanucleotide d(GCATGC) was investigated in aqueous salt solution. Homonuclear 1H NMR correlation spectroscopy (2D-TOCSY and 2D-NOESY) was used for complete assignments of nonexchangeable protons of the hexamer. The equilibrium reaction constants and thermodynamical parameters of duplex d(GCATGC)2 formation were determined from experimental concentration and temperature dependences of proton chemical shifts of the deoxyhexanucleotide. Distinctive features of the concentration dependences in the range of small concentrations at relatively low temperatures of solution enable one to assume that one single-stranded hexamer sequence forms a compact structure (similar to a hairpin) in aqueous solution. A possible spatial hairpin structure of the hexamer was proposed. Comparative analysis of the experimental and theoretical (using the "nearest neighbor" model) thermodynamical parameters of duplex formation was made.     

Effect of second-sphere coordination 12. Adduct formation behavior of crown ethers with ammine complexes of ruthenium containing a protic ligand of other type than ammine

Adduct formation of pentaammineruthenium complexes involving a different type of protic ligand, such as imidazole, was investigated for a series of crown ethers with different ring size. Changes in redox potential and in absorption spectra of the complex were measured on addition of crown ether to the complex solution. The magnitude of the change in both properties is dependent on the ring size of crown ethers. ¹H-NMR spectra of the complex were measured in the presence of crown ethers in order to elucidate hydrogen bonding sites. The chemical shifts of NH proton of imidazol and ammine protons were measured at various concentrations of crown ethers. Adduct formation was discussed based on the features of dependences of those chemical shifts on crown ether concentration.     

Heteroassociation of antibiotic norfloxacin with aromatic vitamins in aqueous solution

The heteroassociation of the antibacterial antibiotic norfloxacin with aromatic vitamins nicotineamide and flavin mononucleotide in aqueous solution has been studied by 1H NMR spectroscopy (503 MHz). Equilibrium constants, induced proton chemical shifts, and the thermodynamic parameters (deltaH, deltaS) of the heteroassociation of molecules were determined from the concentration and temperature dependences of chemical shifts of protons of interacting aromatic molecules. An analysis of the results indicates the formation of heterocomplexes between the molecules of the vitamins and norfloxacin, which is caused by stacking interactions between aromatic chromophores and an additional intermolecular hydrogen bond in the norfloxacin-nicotinamide system. Based on the analysis of induced chemical shifts of protons of molecules, the most probable spatial structures 1:1 of norfloxacin-flavin mononucleitide and norfloxacin-nicotinamide heterocomolexes were determined by the methods of molecular modeling using the X-PLOR program.     

d-CpCpGpG and d-GpGpCpC self-complementary duplexes: Nmr studies of the helix-coil transition

We have monitored the helix-coil transition of the self-complementary d-CpCpGpG and d-GpGpCpC sequences (20mM strand concentration) at the base pairs, sugar rings, and backbone phosphates by 360-MHz proton and 145.7-MHz phosphorus nmr spectroscopy in 0.1M phosphate solution between 5 and 95°C. The guanine 1-imino Watson-Crick hydrogen-bonded protons, characteristic of the duplex state, are observed below 10°C, with solvent exchange occurring by transient opening of the tetranucleotide duplexes. The cytosine 4-amino Watson-Crick hydrogen-bonded protons resonate 1.5 ppm downfield from the exposed protons at the same position in the tetranucleotide duplexes, with slow exchange indicative of restricted rotation about the C-N bond below 15°C. The guanine 2-amino exchangeable protons in the tetranucleotide sequence exhibit very broad resonances at low temperatures and narrow average resonances above 20°C, corresponding to intermediate and fast rotation about the C-N bond, respectively. Solvent exchange is slower at the amino protons compared to the imino protons since the latter broaden out above 10°C. The well-resolved nonexchangeable base proton chemical shifts exhibit helix-coil transition midpoints between 37 and 42°C. The transition midpoints and the temperature dependence of the chemical shifts at low temperatures were utilized to differentiate between resonances located at the terminal and internal base pairs while the H-5 and H-6 doublets of individual cytosines were related by spin decoupling studies. For each tetranucleotide duplex, the cytosine H-5 resonances exhibit the largest chemical shift change associated with the helix-coil transition, a result predicted from calculations based on nearest-neighbor atomic diamagnetic anisotropy and ring current contributions for a B-DNA duplex. There is reasonable agreement between experimental and calculated chemical shift changes for the helix-coil transition at the internal base pairs but the experimental shifts exceed the calculated values at the terminal base pairs due to end-to-end aggregation at low temperatures. Since the guanine H-8 resonances of the CpCpGpG and d-CpCpGpG sequences exhibit upfield shifts of 0.6–0.8 and <0.1 ppm, respectively, on duplex formation, these RNA and DNA tetranucleotides with the same sequence must adopt different base-pair overlap geometries. The large chemical shift changes associated with duplex formation at the sugar H-1′ triplets are not detected at the other sugar protons and emphasize the contribution of the attached base at the 1′ position. The coupling sum between the H-1′ and the H-2′ and H-2″ protons equals 15–17 Hz at all four sugar rings for the d-CpCpGpG and d-GpGpCpC duplexes (25°C), consistent with a C-3′ exo sugar ring pucker for the deoxytetranucleotides in solution. The temperature dependent phosphate chemical shifts monitor changes in the ω,ω′ angles about the O-P backbone bonds, in contrast to the base-pair proton chemical shifts, which monitor stacking interactions.