Hairpin formation of d(CGCG-TA-CGCG), d(CGCG-TG-CGCG) and their cytosine methylated analogs

Hairpin formations of decamers d(CGCG-TA-CGCG), d(CGCG-TG-CGCG), and their m5dC analogs are evidenced by the existence of biphasic absorbance melting profiles in which the lower transition temperature increases with increasing oligomer concentration, whereas the higher melting temperature is concentration independent. The corresponding temperature dependent CD intensity at 285 nm exhibits a maximum around 55 degrees C. These observations are consistent with the interpretation that the lower temperature transition corresponds to the duplex to hairpin transformation while the melting of hairpins into single strands constitutes the higher temperature transition. The CD spectrum of the hairpin conformation appears to be characterized by a couplet with nearly equal positive and negative intensities at 285 and 255 nm, respectively, while a significantly smaller intensity at 285 nm is apparent for the duplex form. The hairpin conformation is suspected to contain a two-nucleotide loop. Titrations with NaCl further suggest that, in contrast to the TA sequence, the TG sequence with wobble base pairing favors Z formation under high salt conditions.     

The Duplex-Harpin Conformational Transition of d(CGCGCGATCGCGCG) and d(CGCGCGTACGCGCG): A Thermodynamic and Kinetic Study

Abstract

We have studied the duplex-hairpin conformational transition in two perfectly palindromic sequences, d(CGCGCGATCGCGCG)(I) and d(CGCGCGTACGCGCG)(II), by means of UV-melting, electrophoretic and T-jump experiments. Both tetradecamers exhibit biphasic thermal profiles. The lower temperature transition is concentration dependent whereas the higher temperature transition is not. The former transition has been characterized by gel electrophoresis and shows two distinct bands, whose intensity depends on temperature. This behavior is due to the occurrence of a slow premelting interconversion between the duplex and hairpin forms in both tetradecamers. The kinetics of hairpin formation from the duplex is studied by T-jump experiments. Relaxation spectra are well reproduced by a single relaxation time with rate constants characterized by a high temperature coefficient. In 10 mM NaCl, the duplex-hairpin conversion of I is characterized by an apparent activation energy of 96 ± 6 kcal/mol, a value rather close to the expected denaturation enthalpy. In 1 mM NaCl a value slightly lower has been obtained. The rate of duplex-hairpin interconversion has been found to decrease as the salt concentration is raised. These data suggest that the transformation from the duplex to the hairpin form should imply a transition state with a simultaneous breaking of most base pairs, if not total strand separation.     

Salt-dependent conformational transitions in the self-complementary deoxydodecanucleotide d(CGCAATTCGCG): Evidence for hairpin formation

Differential scanning calorimetry (DSC), temperature-dependent uv-absorption spectroscopy, and temperature-dependent CD were used to monitor and characterize the salt-dependent, thermally induced structural transitions in the deoxydodecanucleotide d(CGCGAATTCGCG). At the high oligomer concentrations required for DSC, the calorimetric scans revealed a single, monophasic transition curve at all salt concentrations. Based on previous nmr melting studies under similar conditions, we conclude that these monophasic transitions correspond to the cooperative duplex-to-single-strand conversion of the dodecamer. By contrast, at the lower oligomer concentrations used for the spectroscopic studies, the shapes of the uv and CD melting curves were found to depend on the concentration of the added salt. At high salt (≥0.1M Na⁺), a single, monophasic transition curve was observed. At lower salt (?0.01M Na⁺), the CD and uv melting curves exhibit biphasic behavior. Based on the concentration dependence, the enthalpy, and the cooperativity of each transition in the biphasic curve, we conclude that at low salt and low oligomer concentrations, the dodecamer melts in a sequential manner involving initial disruption of a duplex structure and subsequent disruption of a hairpin structure.     

Kinetic and thermodynamic characterization of DNA duplex–hairpin interconversion for two DNA decamers: d(CAACGGGTTG) and d(CAACCCGTTG)

The duplex–hairpin interconversion of two DNA decamers, d(CAACGGGTTG) and d(CAACCCGTTG), has been characterized thermodynamically and kinetically by using uv-melting and nmr relaxation methods. Separately, each decamer shows slow exchange between hairpin and duplex conformations. The hairpin conformations have melting points of 47 and 50°C, respectively, and exhibit similar thermodynamic stabilities. The enthalpies of duplex formation, measured by nmr, were found to be very similar (ΔH_DH = 26 ± 3 kcal/mole) for both decanters at low salt concentrations (< 50 mM NaCl). However, as the salt concentration was increased the behavior of ΔH_DH, and kinetics is significantly different for each decamer. The d(CAACGGGTTG) decamer forms a duplex containing two central G·G mismatches at high salt and DNA concentration. Based upon the measurement of high interconversion activation energies and a decrease in hairpin formation rate with increasing salt, the interconversion between hairpin and duplex was concluded to proceed by complete strand dissociation. In contrast, the d(CAAC-CCGTTG) decamer was determined to form a duplex with two centrally located C·C mismatches at pH values less than 6.2, consistent with the formation of a hemiprotonated C⁺·C mismatch. At pH values greater than 6.4, the hairpin–duplex equilibrium is almost completely shifted toward the hairpin conformation at DNA concentrations of 0.5–7.0 mM and salt concentrations of 10–100 mM. The interconversion of duplex and hairpin conformations was ascertained by means of both kinetic and thermodynamic measurements to proceed by a slightly different mechanism than its complementary decamer. Although the interconversion proceeds by complete strand separation as suggested by high duplex-hairpin interconversion activation enthalpies, the increasing hairpin formation rate with increasing ionic strength as well as the ΔH_DH, dependence on sail indicate that an intermediate internally bulged duplex (no C⁺·C formation) is stabilized by increasing ionic strength. These data support an interconversion mechanism where an intermediate internally bulged duplex may be the rate limiting step before strand separation. © 1995 John Wiley & Sons, Inc.     

Energetics of the hairpin to mismatched duplex transition of d(GCCGCAGC) on NaCl solution

We report Potential of Mean Force studies to describe the relative thermodynamic stabilities of d(GCCGCAGC) in a mismatched duplex and a hairpin monomer conformation in NaCl solution. The PMF calculations are combined with previous molecular mechanics and normal mode analysis in order to estimate the role of different components of the free energy in determining the relative stability of the duplex and hairpin structures. The high entropy associated with the loop region and the lack of minor groove phosphate-phosphate interactions in the hairpin compete against the gain in enthalpic contribution to the free energy due to base pairing in the mismatched duplex. The combined free energy calculations show that the hairpin is the most stable conformation at low salt and that a hairpin to duplex transition takes place at approximately 0.47 M NaCl. In addition, we studied the hairpin to partially stacked single helical conformation equilibrium at low salt. We found a small variation in transition temperature in salt concentration, delta Tm/delta log10(cs) approximately 2-3 degrees K/decade, in contrast to the duplex to hairpin or duplex to partially stacked single helix transition where the transition temperature exhibited marked dependence on salt concentration. This is in qualitative agreement with experimental data. Based on the Potential of Mean Force free energy calculation, the order of relative stability of the three-conformations studied varies with salt concentration. We observed the following orders of stability: stacked single helix greater than hairpin greater than duplex for cs less than 0.77 M NaCl; single helix greater than duplex greater than hairpin for 0.77 less than Cs less than 2.1 M; and duplex greater than hairpin greater than single strand for cs greater than 2.1 M. From the calculated PMF free energy curves in the NaCl concentration range, 0.012 less than cs less than 5.0 M, we can assign upper and lower bounds for the non-ionic differences in free energy between the duplex, hairpin, and stacked single helical states (at standard conditions: cs = 1.0 M, T = 25 degrees C, and 1 M oligomer concentration). We found that for delta G duplex single helix = G duplex - 2 x G single helix less than -7.38 Kcal/mol, the single helix is the least stable state. For the duplex-to-hairpin free energy difference in the range, -1.87 less than delta G duplex-hairpin less than 0.03 Kcal/mol, there will always be a salt-induced hairpin-to-duplex transition for 0.01 less than cs less than 1.6 M NaCl. If delta G duplex-hairpin less than -1.87, the duplex is always more stable than the hairpin; and for delta G duplex-hairpin greater than Kcal/mol, the hairpin state is always more stable than the duplex, for all salt concentrations.     

Structural polymorphism exhibited by a quasipalindrome present in the locus control region (LCR) of the human beta-globin gene cluster

Structural polymorphism of DNA is a widely accepted property. A simple addition to this perception has been our recent finding, where a single nucleotide polymorphism (SNP) site present in a quasipalindromic sequence of β-globin LCR exhibited a hairpin-duplex equilibrium. Our current studies explore that secondary structures adopted by individual complementary strands compete with formation of a perfect duplex. Using gel-electrophoresis, ultraviolet (UV)-thermal denaturation, circular dichroism (CD) techniques, we have demonstrated the structural transitions within a perfect duplex containing 11 bp quasipalindromic stretch (TGGGG(G/C)CCCCA), to hairpins and bulge duplex forms. The extended version of the 11 bp duplex, flanked by 5 bp on both sides also demonstrated conformational equilibrium between duplex and hairpin species. Gel-electrophoresis confirms that the duplex coexists with hairpin and bulge duplex/cruciform species. Further, in CD spectra of duplexes, presence of two overlapping positive peaks at 265 and 285 nm suggest the features of A- as well as B-type DNA conformation and show oligomer concentration dependence, manifested in A → B transition. This indicates the possibility of an architectural switching at quasipalindromic region between linear duplex to a cruciform structure. Such DNA structural variations are likely to be found in the mechanics of molecular recognition and manipulation by proteins.     

Studies on formation and stability of the d[G(AG)5]* d[G(AG)5]·d[C(TC)5] and d[G(TG)5]* d[G(AG)5]· d[C(TC)5] triple helices

We have targeted the d[G(AG)₅] · d[C(TC)₅] duplex for triplex formation at neutral pH with either d[G(AG)₅] or d[G(TG)₅]. Using a combination of gel electrophoresis, uv and CD spectra, mixing and melting curves, along with DNase I digestion studies, we have investigated the stability of the 2:1 pur*pur · pyr triplex, d[G(AG)₅] * d[G(AG)₅] · d[C(TC)₅], in the presence of MgCl₂. This triplex melts in a monophasic fashion at the same temperature as the underlying duplex. Although the uv spectrum changes little upon binding of the second purine strand, the CD spectrum shows significant changes in the wavelength range 200–230 nm and about a 7 nm shift in the positive band near 270 nm. In contrast, the 1:1:1 pur/pyr*pur · pyr triplex, d[G(TG)₅] * d[G(AG)₅] · d[C(TC)₅], is considerably less stable thermally, melting at a much lower temperature than the underlying duplex, and possesses a CD spectrum that is entirely negative from 200 to 300 nm. Ethidium bromide undergoes a strong fluorescence enhancement upon binding to each of these triplexes, and significantly stabilizes the pur/pyr*pur · pyr triplex. The uv melting and differential scanning calorimetry analysis of the alternating sequence duplex and pur*pur · pyr triplex shows that they are lower in thermodynamic stability than the corresponding 10-mer d(G₃A₄G₃) · d(C₃T₄C₃) duplex and its pur*pur · pyr triplex under identical solution conditions. © 1997 John Wiley & Sons, Inc.     

The duplex-hairpin conformational transition of d(CGCGCGATCGCGCG) and d(CGCGCGTACGCGCG): a thermodynamic and kinetic study

We have studied the duplex-hairpin conformational transition in two perfectly palindromic sequences, d(CGCGCGATCGCGCG)(I) and d(CGCGCGTACGCGCG)(II), by means of UV-melting, electrophoretic and T-jump experiments. Both tetradecamers exhibit biphasic thermal profiles. The lower temperature transition is concentration dependent whereas the higher temperature transition is not. The former transition has been characterized by gel electrophoresis and shows two distinct bands, whose intensity depends on temperature. This behavior is due to the occurrence of a slow premelting interconversion between the duplex and hairpin forms in both tetradecamers. The kinetics of hairpin formation from the duplex is studied by T-jump experiments. Relaxation spectra are well reproduced by a single relaxation time with rate constants characterized by a high temperature coefficient. In 10 mM NaCl, the duplex-hairpin conversion of I is characterized by an apparent activation energy of 96 +/- 6 kcal/mol, a value rather close to the expected denaturation enthalpy. In 1 mM NaCl a value slightly lower has been obtained. The rate of duplex-hairpin interconversion has been found to decrease as the salt concentration is raised. These data suggest that the transformation from the duplex to the hairpin form should imply a transition state with a simultaneous breaking of most base pairs, if not total strand separation.     

Thermodynamic behaviour of the heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG) forming B and Z hairpins in aqueous solution.

UV and CD data of the partially self-complementary heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG), obtained as a function of temperature, salt and strand concentration, show that: at low NaCl and strand concentration the oligomer exhibits, on increasing the temperature, a biphasic thermal profile which is indicative of two structural transitions, from dimeric duplex to hairpin and from hairpin to coil; the loop stabilizes enthalpically both B and Z hairpin structures with respect to the corresponding unconstrained hexamer d(CGCGCG) by a few Kcal/mol; the oligomer undergoes a B-Z transition which appears to be complete, at 0 degree C, when induced by NaClO4; by contrast the B-Z transition induced by NaCl does not reach completeness even at salt saturation. The independence of the denaturation temperature, at high salt conditions, on the oligomer concentration indicates that the Z structure is present also in the hairpin.     

Hairpin Formation In d-AAGCTTAAGCTT Under High Salt Conditions Shows Unusual Properties

Abstract

The hairpin-duplex equillibria of the dodecamer d-AAGCTTAAGCTT and interaction of the duplex form with a pentapeptide, KGWGK, has been studied. UV thermal transitions are monophasic at low salt but biphasic at higher salt concentrations. At 10^?5M or less oligomer concentration biphasic melting curves persist till 900 mM NaCl. The d(Tm)/d log(Na⁺) for the duplex form is 12 °C and for the hairpin is 18 °C. The ΔH and ΔS values for duplex formation are low(-25 Kcal/mole and—59 Cal/mole respectively). KGWGK binds to the duplex form with a binding constant K = 3.4×10⁵M^?1measured from fluorescence quenching of tryptophan. These unusual results are markedly different from that reported for d-AGATCT- AGATCT (Biochemistry 31, 6241–6245) and are discussed in ternis of sequence dependence of loop folding and cruciform extrusion pathway of hairpin formation.     

Influence of cationic molecules on the hairpin to duplex equilibria of self-complementary DNA and RNA oligonucleotides

A self-complementary nucleotide sequence can form both a unimolecular hairpin and a bimolecular duplex. In this study, the secondary structures of the self-complementary DNA and RNA oligonucleotides with different sequences and lengths were investigated under various solution conditions by gel electrophoresis, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy and a ultraviolet (UV) melting analysis. The DNA sequences tended to adopt a hairpin conformation at low cation concentrations, but a bimolecular duplex was preferentially formed at an elevated cationic strength. On the other hand, fully matched RNA sequences adopted a bimolecular duplex regardless of the cation concentration. The thermal melting experiments indicated a greater change in the melting temperature of the bimolecular duplexes (by ~20°C) than that of the hairpin (by ~10°C) by increasing the NaCl concentration from 10 mM to 1 M. Hairpin formations were also observed for the palindrome DNA sequences derived from Escherichia coli, but association of the complementary palindrome sequences was observed when spermine, one of the major cationic molecules in a cell, existed at the physiological concentration. The results indicate the role of cations for shifting the structural equilibrium toward a nucleotide assembly and implicate nucleotide structures in cells.     

Deoxydodecanucleotide heteroduplex d(TTTTATAATAAA). d(TTTATTATAAAA) containing the promoter Pribnow sequence TATAAT. I. Double-helix stability by UV spectrophotometry and calorimetry

Thermally induced structural transition in the d(TTTTATAATAAA) d(TTTATTATAAAA) heteroduplex is characterized by UV-spectroscopy and differential scanning calorimetry. At low salt (less than 0.1 M) the occurrence of a cooperative transition in the lower temperature range, followed by a broad transition connected with small increase in absorbance is observed. At high salt (greater than or equal to 0.2 M) a single, monophasic transition appears. Linear dependence of the latter on log of salt concentration (dTm:dlogM = 14.2 degrees C) and of 1/Tm on log of oligomer concentration [derived therefrom delta H (v.H.) = 77.1 kcal/mole (duplex)] allows relating it to the melting of the heteroduplex helix. The non-cooperative transition, independent of oligomer concentration and similar to that of the single chain, was attributed to melting of short hairpin helices upon heteroduplex dissociation. Calorimetric enthalpy: 75.6 kcal/mole (duplex) proved significantly lower than predicted from known calorimetric data for poly[d(AT)] and poly d(A) X poly d(T).     

Effect of base-pair sequence on the conformations and thermally induced transitions in oligodeoxyribonucleotides containing only AT base pairs

Tm curves, CD spectra, and kinetics results of the self-complementary DNA dodecamers d(A6T6), d(A3T3A3T3), d(A2T2A2T2A2T2), d(ATATATATATAT), and d(T6A6) demonstrate that the thermal transitions of these oligomers at low salt concentration involve a hairpin intermediate. At high salt concentrations (greater than 0.1 M Na+) only a duplex to denatured-strand transition appears to occur. The temperature and salt-concentration regions of the transitions are very sequence dependent. Alternating-type AT sequences have a lower duplex stability and a greater tendency to form hairpins than sequences containing more nonalternating AT base pairs. Of the two nonalternating sequences, d(T6A6) is significantly less stable than d(A6T6). Both oligomers have CD curves that are very similar to the unusual CD spectrum of poly(dA).poly(dT). The Raman spectra of these two oligomers are also quite similar, but at low temperature, small intensity differences in two backbone modes and three nucleoside vibrations are obtained. The hairpin to duplex transition for the AT dodecamers was examined by salt-jump kinetics measurements. The transition is faster than transitions for palindromic-sequence oligomers containing terminal GC base pairs. Stopped-flow kinetics studies indicate that the transition is second order and has a relatively low activation energy. The reaction rate increases with increasing ionic strength. These results are consistent with a three-step mechanism for the hairpin to duplex reaction: (i) fraying of the hairpin oligomers' terminal base pairs, (ii) a rate-determining bimolecular step involving formation of a cruciform-type intermediate from two hairpin oligomers with open terminal base pairs, and (iii) base-pair migration and formation in the intermediate to give the duplex.     

Conformation and dynamics of a left-handed Z-DNA hairpin: studies of d(CGCGCGTTTTCGCGCG) in solution

The physical properties of the DNA oligomer d(CGCGCGTTTTCGCGCG) in solvents containing 4 M NaClO4 and 0.1 M NaCl were investigated by proton NMR, optical melting, and circular dichroism spectroscopy. Results of these investigations are as follows: (i) The DNA hexadecamer exists as a unimolecular hairpin in either high or low salt. (ii) In high salt the stem region of the hairpin is in the left-handed Z conformation. (iii) In either high or low salt, the duplex stem of the hairpin is stabilized against melting by approximately 40 degrees C compared to the linear core duplex. The added stability of the hairpin is entropic in origin. (iv) In high salt, as the temperature is elevated, the equilibrium structure of the duplex stem of the hairpin shifts from the Z to the B conformation before melting. (v) In low salt, when the DNA duplex exists in the B conformation, attachment of a T4 single-strand loop to one end only slightly decreases (by 14%) the correlation time of the CH5-CH6 interproton vector. In high salt, when the DNA duplex exists in the Z conformation, the correlation time of the CH5-CH6 interproton vector decreases by 51%. Since these viscosity-corrected correlation times are taken to be indicators of duplex motions on the nanosecond time scale, this result directly suggests a larger amplitude of these motions is present in the duplex stem of the hairpin when it exists in the Z conformation.     

Infrared Spectral Studies of the Non Regularly Alternating Purine-Pyrimidine Hexamers d(m5CGGCM5CG), d(CBr8GGCCBr8G) and d(CGCGGC)

Abstract

The oligonucleotides d(m⁵CGGCm⁵CG), d(CBr⁸GGCCBr⁸G) and d(CGCGGC) have been prepared and studied by infrared spectroscopy. The three sequences contain two GC pairs which are out of purine-pyrimidine alternation with the rest of the sequence. From the IR data of the dlm⁵CGGCm^CG) hexamer, it is shown that all of the dG residues adopt a syn conformation. The marker IR bands for the C3′ endo syn conformation are at 1410, 1354, 1320 and 925 cm^?1 whereas those for the C2′ endo and conformation at 1420, 1374 and 890 cm^?1 are clearly absent. This result implies that the two adjacent guanines of the d(m⁵CGGCm⁵CG) sequence are in syn conformation. It is suggested that duplex formation occurs in d(CGCGGC) films and that all of the guanines are in syn conformation. In contrast, the central non-brominated guanine of the dlCBr⁸GGCCBr⁸G) hexamer is found in ami conformation, as expected in a Z type structure of the non-alternating region.     

Melting of Oligodeoxynucleotides with Various Structures

Abstract

Effects of DNA fragments end structures on their melting profiles were studied experimentally and theoretically. We examined melting of hairpins and dumbbells obtained from 62- bp-long linear DNA duplex which is a perfect palindromic sequence. To fit theoretical melting profile to experimental ones additional theoretical parameters were incorporated into the standard statistical mechanical helix-coil transition theory. From comparison theoretical and experimental melting profiles theoretical parameters connected with end- structure effects were evaluated. Analysis revealed the stabilization effect of the hairpin loops and helix ends with respect to DNA duplex melting. Both type of ends make melting these oligodeoxynucleotides more cooperative than predicted by the standard helix-coil transition theory. At low ionic strength ([Na⁺] < 0.04 M) this effect becomes so pronounced that melting of the DNA duplexes 30–40 bp-long conforms to the two state model.

From the analysis experimental data obtained for dumbbell structures loop-weighting factor for single-stranded loop consisting of 132 nucleotides was determined. This parameter decreases 10 times with the ionic strength decreasing by an order of magnitude from 0.2 to 0.02 M Na⁺.     

Laser Photofootprinting of d(C)·d(G)·d(G) Intramolecular Triplex

Abstract

Supercoiling-induced structural transition of the d(C₂₄GC₂₁,) · d(G₂₁CG₂₄) sequence in plasmid DNA in the presence of Mg²⁺ at neutral pH results in alterations of efficiencies of not only single-quantum (pyrimidine[6–4]pyrimidone adducts) but also two-quantum (alkalisensitive lesions of dG residues) photomodifications of nucleoside residues within this sequence. The generation of both types of photoreactions was achieved by the application of high-intensity laser UV radiation (intensity ～ 10¹¹ W/m², pulse duration ～ 10^?8 s, λ= 266 nm) for irradiation of a plasmid DNA The modification extent sufficient for analysis of photoreaction efficiency distributions along both strands of the insert (photofootprinting) was obtained by the action of a single nanosecond pulse of laser UV radiation. The pattern of a laser photofootprinting is consistent with the d(C) · d(G) · d(G) triplex formation in the presence of Mg²⁺ within the insert and shows some details of this triplex structure.     

A comparison of the hairpin stability of the palindromic d(CGCG(A/T)4CGCG) oligonucleotides.

The palindromic deoxyribonucleotides 5'-CGCGA-TATCGCG-3' and 5'-CGCGTTAACGCG-3' have been characterized by 1H NMR spectroscopy. The NMR data identified both B-DNA duplex conformations and hairpin conformations, the latter with loop regions consisting of the four central nucleotides. The resonances of the various conformations were assigned by use of two-dimensional NMR methods. The relative stability of the various conformations was investigated as a function of temperature, ionic strength and nucleotide concentration. The duplexes were found to be stabilized at high ionic strength and at low temperature, while the hairpins were stabilized at low ionic strength and at medium temperature. The thermodynamics of the duplex-hairpin and the hairpin-random coil transitions were examined, and compared to the other two oligonucleotide in the palindromic d(CGCG(A/T)4CGCG) oligonucleotide family. The relative stabilities of the duplex conformations with respect to the random coil conformations are similar for the d(CGCGAATTCGCG), d(CGCGATATCGCG) and d(CGCGTATACGCG) oligonucleotides. The duplex conformation of d(CGCGTTAACGCG) is less stable. The hairpin of d(CGCGTTAACGCG) seems also to be less stable relative to the random coil conformation than in the case of the other oligonucleotides at an equal oligonucleotide concentration. A cruciform intermediate between the duplex and hairpin conformations is suggested to explain some discrepancies observed in this work in case of the d(CGCGTTAACGCG) oligonucleotide. This is similar to what has been reported for the d(CGCGTATACGCG) oligonucleotide.     

2D 1H and 31P NMR Spectra and Distorted A-DNA-Like Duplex Structure of a Phosphorodithioate Oligonucleotide

Abstract

Assignment of the ¹H and ³¹P NMR spectra of a phosphorodithioate modified oligonucleotide decamer duplex, d(CGCTTpS^? ₂AAGCG)₂ (10-mer-S; a site of dithioate substitution is designated with the symbols pS^? ₂), was achieved by two-dimensional homonuclear TOCSY, NOES Y and ¹H-³¹P Pure Absorption phase Constant time (PAC) heteronuclear correlation spectroscopy. In contrast to the parent palindromic decamer sequence (1) which has been shown to exist entirely in the duplex B-DNA conformation under comparable conditions (100 mM KCI), the dithiophosphate analogue forms a hairpin loop. However, the duplex form of the dithioate oligonucleotide can be stabilized at lower temperatures, higher salt and strand concentration. The solution structure of the decamer duplex was calculated by an iterative hybrid relaxation matrix method (MORASS) combined with 2D NOESY-distance restrained molecular dynamics. These backbone modified compounds, potentially attractive antisense oligonucleotide agents, are often assumed to possess similar structure as the parent nucleic acid complex. Importantly, the refined structure of the phosphorodithioate duplex shows a significant deviation from the parent unmodified, phosphoryl duplex. An overall bend and unwinding in the phosphorodithioate duplex is observed. The structural distortion of the phosphorodithioate duplex was confirmed by comparison of helicoidal parameters and groove dimensions. Especially, the helical twists of the phosphorodithioate decamer deviate significantly from the parent phosphoryl decamer. The minor groove width of phosphorodithioate duplex 10-mer-S varies between 8.4 and 13.3 Å which is much wider than those of the parent phosphoryl decamer d(CGCTTAAGCG)₂ (4.2～9.4Å). The larger minor groove width of 10-mer-S duplex contributes to the unwinding of the backbone and indicates that the duplex has an overall A-DNA-like conformation in the region surrounding the dithiophosphate modification.     

Z Helix-coil Transition of d(C-Br8G-C-G-C-Br8G) Studied by CD, 1H-NMR and IR Spectroscopies

Abstract

The conformation of díC-Bi⁸G-C-G-C-Br⁸G) in aqueous solution was studied by CD and ¹H-NMR spectroscopy and in condensed phase by IR spectroscopy. Whether in 0.1 M or 3 M NaCl solution or in film the only double helical structure adopted by brominated d(C-G)₃ oligomer is the Z form. The IR spectrum of the film presents all the characteristic absorptions of the Z conformation and in particular is indicative of a syn conformation for the central guanosine as well as for the brominated one. Imino proton resonances of diC-Bi⁸G-C- G-C-Br⁸G) demonstrating the duplex formation were observed up to 60°C. It is interesting to note that the significant highfield shifts of the dC H₅″ exocyclic sugar protons characteristic of the non exchangeable proton spectra of d(C-G)₃ containing 5-methyl dC residues in the Z form were also detected in the proton spectrum of brominated oligomer. Whereas formation of the Z helix of methylated d(C-G)₃ oligomers dependent on the salt concentration was found to occur via the preliminary formation of a B helix even in 4 M NaCl solution, the Z helix of d(C-Br⁸G-C-G-C-Br⁸G) is obtained directly from the coil form. However, IR data suggest that in the Z form of dlC-Bi⁸G-C-G-C-Bi⁸G), the overlapping of the base planes should be slightly different in comparison with the stacking observed in d(C-G)₃ crystals. The kinetic data (activation energy and lifetime) of the Z helix-coil transition of brominated d(C-G)₃ are compared to those of the B helix-coil transition observed for methylated d(C-G)₃ in 0.1 M NaCl solution while the thermodynamic data of these two reactions (enthalpy and midpoint temperature) are slightly different.