dC-dG alternating oligonucleotides: thermodynamic and kinetic aspects of the B-Z transformation

The alternating cytosine-guanine oligodeoxyribonucleotides (dCdG)n, (dGdC)n, (dCdG)ndC (n = 3,4), (dGdC)7 and dG(dCdG)3 have been studied by UV and CD spectroscopy at different temperatures and NaCl concentrations. The analysis of the melting data, assuming an all-or-none model, reveals that in the B-conformation the 5'G/C3' stacking interactions are enthalpically favoured with respect to the 5'C/G3' one. The CD investigation of the B-Z equilibrium shows that the Z-conformation is enthalpically stabilized, while the B-conformation is entropically favoured, in the range of NaCl concentration considered (1 to 5 M). The kinetic data for the B-Z transformation, obtained with a salt-jump technique for the hexamer (dCdG)3, support a mechanism by which the Watson-Crick hydrogen bonds are broken before the bases flip over separately and eventually stack, reforming the H-bonds, in the new helix.     

Molecular mechanical studies on left- and right-handed B-DNA

Molecular mechanical simulations on base-paired deoxyhexanucleoside phosphates, (dAdT)₃ · (dAdT)₃, (dTdA)₃ · (dTdA)₃, (dGdC)₃ · (dGdC)₃, and (dCdG)₃ · (dCdG)₃, have been carried out to assess their energetic stabilities in left- and right-handed forms. These hexamers have also been simulated with alternating sugar-puckering profiles with the combinations (purine : C2′-endo–pyrimidine : C3′-endo) and (purine : C3′-endo–pyrimidine C2′-endo). The right-handed models have been found to be the energetically most stable structures and the left-handed structures are significantly destabilized. This instability has been rationalized in terms of competition between stabilizing stacking interactions on one hand, and distortions in the bond angles and torsion angles in the sugar-phosphate backbone on the other.     

Effect of Flanking Sequences on the Right- to Left- Handed Transition of a (dA-dT)n Tract in Supercoiled DNA

Abstract

Alternating (dA-dT)_n sequences in supercoiled DNA may undergo a transition to a left-handed conformation in the presence of Ni²⁺ ions and high NaCl concentration (Nejedlý, K., Klysik, J. and Pale?ek, E, FEBS Lett. 243, 313–317 (1989)). In this work we have found that ionic conditions necessary for the B-to-Z transition are strongly dependent on the sequences flanking the (dA-dT)_n tract. In particular, the presence of 5′- homopyrimidine (C3) and 3′-homopurine (G4) blocks adjacent to the tract were found to facilitate the transition to the left- handed form. Within a constant sequence context it was found that the ionic strength required to promote the transition was inversely proportional to the length of the (dA-dT) _n sequence.     

Temperature-dependent reversible transition of poly(dCdG) · poly(DcdG) in ethanolic and methanolic solutions

Using CD we investigated the transitions of poly(dCdG) · poly(dGdC) from B-to-Z form and from Z-to-Z′ form. We have found experimental conditions that allow the cooperative transition to occur as a function of temperature in ethanolic solutions. The transition is reversible and can be repeated as often as desired. There is no evidence of strand separation during the cooperative transition as monitored by absorbance. For purposes of calculation, we have assumed a two-state model for the B-to-Z transition, although the data indicate that such a model is too simplistic. The calculations allow the estimation of the change in enthalpy per mole of cooperative unit for the transition as a function of ethanol concentration. The values range from ±140 to ±200 kcal/mol for ethanol concentrations between 10 and 20%. Investigations of the noncooperative Z-to-Z′ transition show that it is a reversible two-state transition. The different forms of poly(dCdG) · poly(dGdC) give no scattering contributions to the CD as shown by fluorescent-detected CD or fluorscat techniques. This indicates that the CD spectra are true spectra, and contain no contributions from differential scattering of the polynucleotide. This is particularly significant in the case of the Z′ form, since it exists at high ethanol concentrations (80%) where condensation of polynucleotides can provide large contributions to the CD spectra. Analogous investigations using methanol show that the two transitions also occur, but the final Z′ form in methanol is qualitatively different from the ethanol form.     

B,Z Conformations and Mechanism of the Z-B-Coil Transitions of the Self-Complementary Deoxy-hexanucleotide d(C-G-m5C-G-C-G) by 1H-NMR and CD Spectroscopy

Abstract

The helical structures of d(C-G-m⁵C-G-C-G) were studied in aqueous solution at various salt concentrations and temperatures by CD and ¹H-NMR spectroscopy. At room temperature only the B form is observed in 0.1 M NaCl whereas the B and Z forms are simultaneously present in 1.8 M NaCl. At high salt concentration (4 M NaCl) the Z form is largely predominant (> 95%). The Z form proton resonances were assigned by using the polarisation transfer method (between B and Z at 1.8 M NaCl) and by proton-proton decoupling (at high salt concentration).

The Z-B-Coil transitions were studied as a function of temperature with the 1.8 M NaCl solution. At high temperature (95°C) only the coil form (S) is present. Below 55°C the coil proportion is negligible, and the B-Z exchange is slow. The disappearance of the coil gives rise at first to the B form and on lowering the temperature the Z proportion increases to the detriment of the B form. Proton linewidth, relaxation and polarisation transfer studies confirm the conclusion in the previous report on d(m⁵C-G-C-G-m⁵C-G) (Tran-Dinh et al Biochemistry 1984 in the press) that Z exchanges only with B whereas the latter also exchanges with S,Z ? B ? S. The present data show that even at high salt concentration where only the Z form of d(C-G-m⁵C-G-C-G) is observed the Z-S transition also passes through the B form as an intermediate stage. The B-Z transition takes place when the Watson-Crick hydrogen bonds are firmly maintained and is greatly favoured when there are three hydrogen bonds between the base-pairs.     

Strong Patterns in Homooligomer Tracts Occurrences in Non-Coding and in Potential Regulatory Sites in Eukaryotic Genomes

Abstract

Previous studies of the dinucleotides flanking both the 5′ and 3′ ends of homooligomer tracts have shown that some flanks are consistently preferred over others (1,2). In the first preferred group, the homooligomer tracts are flanked by the same nucleotide and/or the complementary nucleotides, e.g., ATA_n, TTA_n, CCG_n, where n=2–5. Runs flanked by nucleotides with which they cannot base pair are distinctly disfavored. (In this group A/T_n are flanked by C and/or G; G_n/C_n are flanked by A/T, e.g., CGA_n, T_nGG, G., AT). The frequencies of runs flanked by AorT, and G or C (“mixed” group) are as expected. Here we seek the origin of this effect and its relevance to protein-DNA interactions. Surprisingly, within the first group, runs flanked by their complements with a pyrimidine-purine junction (e.g., TTA_n, C_nGG) are greatly preferred. The frequencies of their purine-pyrimidine junction mirror-images is just as expected. This effect, as well as additional ones enumerated below, is seen universally in eukaryotes and in prokaryotes, although it is stronger in the former. Detailed analysis of regulatory regions shows these strong trends, particularly in GC sequences. The potential relationship to DNA conformation and DNA-protein interaction is discussed.     

General Nearest Neighbor Preferences in G/C Oligomers Interrupted by A/T: Correlation with DNA Structure

Abstract

The frequencies of occurrence of the 5′ and 3′ nearest neighbor doublets of oligonucleotides containing (G/C) and (A/T) blocks show strong trends. Specifically, the following trends are observed. Given a (G/C)_n (A/T)_m oligomer (where G/C)_n indicates a sequence of length n composed solely of Gs and/or Cs and (A/T)_mis a sequence of length m composed solely of As and/or Ts, and n=3,2,1; m = 1,2,3) and a (G/C)₂ doublet, (G/C)_n (A/T)_m (G/C)_n > (G/C)_n+2 (A/T)_m- That is the (G/C)₂ doublet is preferentially located 3′ of the oligomer, enclosing the (A/T)_m stretch. The trends are strongest for n=3, m= 1 and gradually weaken as the size of the (G/C)_n block decreases (with a concomitant increase of (A/T)_m). (A/T)₂ nearest neighbor flank preferentially encloses the (G/C)_n block (to produce (A/T) ₂(G/C) _n (A/T)_m). The (A/T)₂ flank trends are weaker than the (G/C)₂ flank ones. The (A/T)₂ flank trends also decrease in strength as the size of the (G/C)_n block decreases. The statistical significance of these trends in eukaryotes is very high. A possible correlation with DNA structural parameters, in particular groove geometry, is discussed.     

The Interface Between an Alternating CG Motif and a Random Sequence Motif Displays Altered Nuclease Activity

Abstract

Previously we described the B-Z junctions produced in oligomers containing (5meCG)₄ segments in the presence of 5.0 M NaCl or 50 uM Co(NH₃)₆ ⁺³ [Sheardy, R.D. & Winkle, S.A., Biochemistry 28, 720–725 (1989); Winkle, S. A., Aloyo, M.C., Morales, N., Zambrano, T.Y. & Sheardy, R.D., Biochemistry 30, 10601–10606 (1991)]. The circular dichroism spectra of an analogous unmethylated oligomer containing (CG)₄, termed BZ-IV, in 5.0 M NaCl and in 50 uM CO(NH₃)⁺³ suggest, however, that this oligomer does not form a B-Z hybrid. BZ-IV possesses Hha I sites (CGCG) in the (CG)₄ segment and an Mbo I site (GATC) at the terminus of the (CG)₄ segment BZ-IV is equally digestible in the presence and absence of cobalt hexamine by Hha I, further indicating that the structure of BZ-IV is fully B-like under these conditions. The Mbo I cleavage site at the juncture between the (CG)₄ segment and the adjacent random segment displays enhanced cleavage by both Mbo I and its isoschizomer Sau3A I in the presence of cobalt hexamine. In addition, exonuclease IH digestion of BZ-IV is inhibited at this juncture. Actinomycin inhibits Mbo I activity in the presence of cobalt hexamine but not in the absence. Together, these results suggest that enzymes recognize the interfaces of (CG)n and adjacent random sequences as altered substrates even in the absence of a B-Z junction formation.     

On the absolute configuration of 19′-hexanoyloxyfucoxanthin

The esterifying C₆-acid in 19′-hexanoyloxyfucoxanthin has been identified as n-hexanoic acid by GLC of the methyl ester. Ozonolysis of 19′-n-hexanoyloxyfucoxanthin 3-benzoate provided the n-hexanoyloxy derivative of the allenic ketone produced from fucoxanthin 3-benzoate. NMR and CD correlation of the ozonolysis products and NMR of the native carotenoids provided the basis for assignment of the same absolute configuration of the 19′-n-hexanoyloxy derivative (3S, 5R, 6S, 3′S, 5′R, 6′S) as for fucoxanthin. Biosynthetic implications are considered. CD data for 19′-n-hexanoyloxyfucoxanthin, fucoxanthin and some derivatives thereof are reported. Previously unreported minor carotenoids in Coccolithus huxleyi were diadinoxanthin and 3′-desacetyl 19′-n-hexanoyloxy-fucoxanthin.     

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.     

Polarized Raman scattering from oriented single microcrystals of d(A5T5)2 and d(pTpT)

Polarized Raman spectra have been obtained from single microcrystals of the duplex of the decamer d(A₅T₅)₂ using a Raman microscope. This is the first report of Raman spectra from a crystal of a deoxyoligomer that contains only long, nonalternating sequences of adenine and thymine. Sequences containing d(A)_n and d(T)_n are of interest in view of recent suggestions that they induce bends in DNA and that they might exist in a nonstandard B-conformation. Polarized Raman spectra of a crystal of d(pTpT) have also been obtained. Both crystals display Raman bands whose intensities are very sensitive to the orientation of the crystal with respect to the direction of polarization of the incident laser beam. These spectra indicate that the helical axes of the oligonucleotides are parallel to the long axes of the crystals and that the d(A₅T₅)₂ is not appreciably bent in the crystal. The Raman spectrum from the d(pTpT) crystal indicates that all of the furanose ring puckers are in a C2′-endo configuration since only the C2′-endo marker band at 835 ± 5 cm^?1 is present. Crystals of d(A₅T₅)₂ show measurable Raman intensities in both the 838- and 816-cm^?1 bands. This indicates the presence of both the C2′-endo and C3′-endo, or possibly other non-C2′-endo, furanose conformations. The 816-cm^?1 band is weak so that only a small fraction of the residues are estimated to be in the non-C2′-endo conformation. In both the d(pTpT) and d(A₅T₅)₂ crystals the intensity of the bands due to vibrations of the backbone show only a small dependence on orientation of the crystals. This result is explained by the low symmetry of the puckered sugar rings. It is concluded that Raman spectra obtained from oligonucleotide crystals in which the orientation of the crystal axes to the laser polarization is not carefully controlled may contain intensity artifacts that are due to polarization effects.     

Methylated Polyl(L-lysine): Conformational Effects and Interactions with Polynucleotides

Abstract

Methylated lysine, arginine and histidine residues are found in a number of proteins (for example, histones, non-histone chromosomal proteins, ribosomal proteins, calmodulin, cytochrome C, etc.). We are studying the effects of methylation on the conformations of poly(lysine) and of the effects of methylation of poly(lysine) and poly(arginine) on interactions with polynucleotides. The conformational properties of e-amino-methylated poly(lysine) differ from those of unmodified poly(lysine). Methylation increases resistance to thermally- induced and NaCl-induced changes in the CD spectrum. Guanidinium chloride increases (proportional to the degree of methylation) the extent of approach to the conformation in dispute as to its being a random coil or an extended helix. Methylation enhances aggregation in the helix-inducing solvent 0.5 M Ca(ClO₄)₂. With increasing methylation of poly(lysine), the conformation in dodecyl sulfate changes from β, to 50% α, to random coil at the maximum methylation.

Increasing methylation of poly(lysine) weakens the interaction with polynucleotides in respect to dissociation by salt, linearly with methyl content. Complexes of (dAdT)_n·(dAdT)_n with the polypeptides are increasingly stabilized to heat denaturation by progressive methylation. However, with a series of synthetic double-stranded RNA's and DNA's a more complex situation exists, Tm increasing or decreasing, depending on the base composition, sequence and type of sugar. Methylation of poly(lysine) and poly(arginine) can have opposite effects on Tm based on results with complexes with (dI)_n·(dC)_n. Methylated poly(lysine) affects the CD spectrum of polynucleotides, in a manner dependent on base composition and sequence. In some cases large positive or negative ψ-spectra are induced, which, in the case of (dGdC)_n·(dGdC)_n, can be positive or negative depending on the degree of methylation of the polypeptide and the salt concentration.

It is suggested that the biological effects of methylated proteins may be evoked by salt changes in the cell cycle, and that methylation can affect local interactions with nucleic acids and larger scale structure, and interactions with lipids.     

Interaction of (2′ -5′) and (3′ -5′) Linked 2-Aminoadenylyl-3-aminoadenosines with Polyuridylic Acid

Abstract

2′-5′ and 3′-5′ linked 2-aminoadenylyl-2-aminoadenosines [(2′-5′)n²Apn²A (1) and (3′-5′)n²Apn²A (2)] were synthesized by condensation of 5′-O-monomethoxytrityl-N ² N ⁶-dibenzoyl-2-aminoadenosine and N ²,N ⁶,2′,3′-O-tetrabenzoyl-2-aminoadenosine 5′-phosphate using dicyclohexylcarbodiimide (DCC). The conformational properties of these dimers 1 and 2 were examined by UV, NMR and CD spectroscopy. The results reveal that the 2′-5′-isomer 1 takes a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′-isomer 2. Interactions of 1 and 2 with polyuridylic acid (Poly (U)) were also examined by Tm, mixing curves, UV and CD spectra. Both the dinucleoside isomers 1 and 2 formed a complex of 1 : 2 stoichiometry with poly(U), which was much more stable than that of the corresponding ApA isomer     

Synthesis of 2′,3′-Dideoxy-2′-fluoro-3′-thioarabinothymidine and Its 3′-Phosphoramidite Derivative

Abstract

An efficient method for the synthesis of 5′-O-monomethoxytrityl-2′,3′-dideoxy-2′-fluoro-3′-thioarabinothymidine [^5′-MMTaraF-T_3′SH, (5)] and its 3′-phosphoramidite derivative (6) suitable for automated incorporation into oligonucleotides, is demonstrated. A key step in the synthesis involves reaction of 5′-O-MMT-2,3′-O-anhydrothymidine (4) (Eleuteri, A.; Reese, C.B.; Song, Q., J. Chem. Soc. Perkin Trans. 1 1996, 2237 pp.) with sodium thioacetate to give ^5′-MMTaraF-T_3′SAc (5) (Elzagheid, M.I.; Mattila, K.; Oivanen, M.; Jones, B.C.N.M.; Cosstick, Lönnberg, H. Eur. J. Org. Chem. 2000, 1987–1991). This nucleoside was then converted to its corresponding phosphoramidite derivative, 6, as described previously ((a) Sun, S.; Yoshida, A.; Piccirilli, J.A. RNA, 1997, 3, 1352–1363; (b) Matulic-Adamic, J.; Beigelman, L. Helvetica Chemica Acta 1999, 82, 2141–2150; (c) Fettes, K.J.; O’Neil, I.; Roberts, S.M.; Cosstick, R. Nucleosides, Nucleotides and Nucl. Acids 2001, 20, 1351–1354).     

B-Z Cooperativity and Kinetics of Poly(dG-m5dC) are Controlled by an Unfavorable B-Z Interface Energy

Abstract

Thermodynamic and kinetic properties of the B-Z transition of poly(dG-m⁵dC) were investigated using polynucleotide samples ranging in length from 11000 to 300 base pairs. Van't Hoff enthalpy values increase with increasing polymer length for the B-Z transition in 0.35 mM MgCl₂, 50 mM NaCl, 5 mM TRIS, pH 8. Rates of the B to Z transition increase with increasing polymer length for a jump of 0 to 3 mM MgCl, in 50 mM Nad, 5 mM TRIS, pH 8. The activation energy of the B to Z transition equals 7.9 ± 0.3 kcal/mol and is length independent Thermodynamic and kinetic data were fit to a model that simulates distribution of B- and Z-form tracts at the midpoint of B-Z equilibrium as a function of polymer length. A cooperative length of 1000 ± 200 base pairs is estimated for the B-Z transition. A direct relationship between rates of the B to Z transition and the square of the van't Hoff enthalpy values of the B-Z transition reflects a dependence of kinetics and cooperativity upon the energy of the nucleation event Faster B to Z transition rates with increasing polymer length can be explained by a mechanism rate limited by nucleation within the polymer instead of the ends.     

Synthesis and Conformational Studies of O 5′, 6-Methanouridine—A New Type of Pyrimidine Cyclonucleoside

Abstract

The 5-oxo-6-methylene-pyrimidine-2,4-dione intermediate (6) that is formed when 5-acetoxy-6-acetoxymethyl-1-β-D-(5-O-acetyl-2,3-O-isopropylidene)-ribofuranosyluracil (5) is treated with sodium hydroxide undergoes cyclization at pH 14 to give 2′,3′-O-isopropylidene-5-hydroxy- O ⁵, 6-methanouridine (8) in good yield. Conversion of 8 into the 5-triflate ester 14 followed by reduction with [(Ph)₃P]₄Pd/Bu₃SnH and deblocking with acetic acid then affords O ^5′, 6-methanouridine (4) Conformational studies (NOE difference spectra, vicinal ¹H-¹³C coupling constants, NOESY and CD spectra, molecular modeling) indicate that the C7-methylene group of 4 projects towards the furanose ring oxygen atom, producing a glycosyl rotation angle of about ? 160°.     

An analysis of the sequence dependence of the structure and energy of A- and B-DNA models using molecular mechannics

Molecular-mechanics calculations have been carried out on the base-paired hexanucleoside pentaphosphates d(TATATA)₂, d(ATATAT)₂, d(A₆)·d(T₆), d(CGCGCG)₂, d(GCGCGC)₂, and d(C₆)·d(G₆) in both A- and B-DNA geometries. The calculated relative energies of these polymers are consistent with the relative stabilities of the polymers found experimentally. In particular, the results of our calculations support the observation that the homopolymer d(A)_n·d(T)_n is more stable in a B-DNA conformation, while the homopolymer d(G)_n·d(C)_n is more stable in an A-DNA conformation. The molecular interactions responsible for these differential stabilities include both inter- and intrastrand base stacking, as well as base–phosphate interactions. While definitive experiments on the heteropolymer stabilities have not yet been carried out, the results of our calculations also suggest a greater stability of the purine-3′,5′-pyrimidine sequence over the pyrimidine-3′,5′-purine sequence in both the A- and B-conformations. The reason for this greater stability lies in the importance of the inherent directionality (5′ → 3′ vs 3′ → 5′) of phosphate–base and base–base interactions. The largest conformation change observed on energy refinement is sugar repuckering, which occurs mainly on pyrimidine-attched sugars and only in the B-DNA geometry. We suggest a molecular mechanism, specifically, differential base–sugar steric interactions involving neighboring sugars, to explain why this repuckering occurs more with d(A₆)·d(T₆) than with other isomers.     

Strong sequence patterns in eukaryotic promoter regions: Potential implications for DNA structure

• 1.1. Analysis of eukaryotic sequences reveals recurring trends in upstream regions. Oligomers composed of (G/C)_n and (A/T)_m blocks are preferentially flanked by (G/C)₂ doublets on their 3' rather than on their 5′ ends, that is (G/C)_nä(A/T)_m(G/C)₂ > (G/C)_n+2(A/T)_m.
• 2.2. These trends are stronger for larger n and smaller m. Additional trends are outlined below.
• 3.3. The trends are correlated with DNA structural parameters, in particular with twist and roll angles.
• 4.4. Generally, the trends hold if the base pair step joining the 5′ (G/C)₂ doublet to the (G/C)_n (A/T)_m oligomer is not undertwisted and is not strongly rolled into the major groove.
• 5.5. Other DNA parameters crucial for DNA-protein interactions are discussed as well.

     

Nucleosides and Nucleotides. 105. DNA Bending in d(A)4 - d(T)4 TRACTS Containing 3-Deazaadenine or 7-Deazaadenine Substituted for Adenine§,1

Abstract

Self-complementary decadeoxyribonucleotides containing 3-deazaadenine or 7-deazaadenine in place of one of the adenine moieties in a parent sequence, d(GAAAATTTTC)_n, have been synthesized. After phosphorylation at their 5′-ends, the decamers were ligated to form multimers, which were analyzed by polyacrylamide gel electrophoresis. The multimers of the decamer containing 3-deazaadenine (3), d(GAA3ATTTTC)_n, showed decreased bending compared with the multimers of d(GAAAATTTTC)_n, while the decamer containing 3-deazaadenine at a different position, d(GAAA3TTTTC)_n, did not show any degree of bending. Also, the properties of migration of the multimers containing 7-deazaadenine in the gel will be discussed.     

Trifurcated (Four-Center) Hydrogen Bond in Solid State Crystal Structure of 5′-Amino-5′-deoxyadenosine p-Toluenesulfonate

Abstract

The crystal structure of 5′-amino-5′-deoxyadenosine (5′-Am.dA) p-toluenesulfonate has been determined by X-ray crystallographic methods. It belongs to the orthorhombic space group P2₁2₁2₁ with a=7.754(3)Å, b=8.065(l)Å and c=32.481(2)Å. This nucleoside shows a syn conformation about the glycosyl bond and C2′-endo-C3′-exo puckering for the ribose sugar. The orientation of N5′ atom is gauche-trans about the exocyclic C4′-C5′ bond. The amino nitrogen N5′ forms a trifurcated hydrogen bond with N3, O9T and 04′ atoms. Adenine bases form A.A.A triplets through hydrogen bonding between N6, N7 and N1 atoms of symmetry related nucleoside molecules.