Assignment of phosphorus-31 and nonexchangeable proton resonances in a symmetrical 14 base pair lac pseudooperator DNA fragment

The 31P chemical shifts of all 13 phosphates and the chemical shifts of nearly all of the non-exchangeable protons of a symmetrical 14 base pair lac pseudooperator DNA fragment have been assigned by regiospecific labeling with oxygen-17 and two-dimensional NMR techniques. At 22 degrees C, 8 of the 13 phosphorus resonances can distinctly be resolved while the remaining 5 resonances occur in two separate overlapping regions. The 31P chemical shifts of this particular 14 base pair oligonucleotide do not follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence the more upfield the 31P resonance occurs, as shown from other 31P assignment studies. Failure of this general rule is believed to be a result of helical distortions that occur along the oligonucleotide double helix, on the basis of the analysis of Callidine [Callidine, C.R. (1982) J. Mol. Biol. 161, 343-352]. Notable exceptions to the phosphate position relationship are 5'-Py-Pu-3' dinucleotide sequences, which resonate at a lower field strength than expected in agreement with similar results as reported by Ott and Eckstein [Ott, J., & Eckstein, F. (1985) Biochemistry 24, 253]. A reasonable correlation exists between 31P chemical shifts values of the 14-mer and the helical twist sum function of Calladine. The most unusual 31P resonance occurs most upfield in the 31P spectrum, which has been assigned to the second phosphate position (5'-GpT-3') from the 5' end. This unusual chemical shift may be the result of the predicted large helical twist angle that occurs at this position in the 14-mer sequence. Further, it is believed that the large helical twist represents a unique structural feature responsible for optimum binding contact between lac repressor protein and this 14-mer lac pseudooperator segment. Assignments of proton resonances were made from two-dimensional 1H-1H nuclear Overhauser effect (NOESY) connectivities in a sequential manner applicable to right-handed B-DNA, in conjunction with two-dimensional homonuclear and heteronuclear J-correlated spectroscopies (1H-1H COSY and 31P-1H HETCOR). Most nonexchangeable base proton and deoxyribose proton (except for some unresolved H4', H5', and H5" protons) resonances were assigned.     

Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.     

Structural and dynamic properties of a fluorouracil-adenine base pair in DNA studied by proton NMR

An oligodeoxynucleotide duplex containing the chemotherapeutic agent 5-fluorouracil (FU) has been constructed by solid phase phosphotriester synthesis and has been studied in solution by proton NMR. In this study, we provide the first structural characterization of a DNA complex containing a FU.A base pair. It has been determined that the 7-mer duplex containing a central FU.A base pair adopts a normal right-handed configuration and the A residue in the FU.A pair is oriented in the normal anticonfiguration giving a Watson-Crick base pair. The significant difference between T.A and FU.A base pairs is dynamic, not structural: the FU.A base pair opens faster than normal base pairs in the oligonucleotide studied. We provide evidence that the FU.A base pair has a significantly enhanced opening rate resulting form decreased stacking of the 5-fluorouracil residue and not from the enhanced acidity of the 5'-fluorouracil imino proton.     

Nuclear magnetic spectra of self complementary decanucleotides in solution; base sequence effect on the chemical shifts of nonexchangeable protons

The aim of this study was to attempt to determine the extent to which the chemical shifts of the nonexchangeable base protons of a DNA helix depend upon the base sequence. We measured the proton NMR spectra of twelve decadeoxynucleotides in order to carry out a "statistical" treatment. In the helices, the chemical shifts were found to be determined within +/- 0.04 ppm, largely by the nearest neighbor residues on the 5'-side, and to a smaller extent by the residue on the 3'-side. The theoretical chemical shift calculations reproduced very well the polymerization shifts measured for H2 protons of adenosines if the electrostatic field effect was taken into account. A fair agreement was also obtained for H8 protons of the adenosine and guanosine residues. However, theory underestimates the polarization effects of the base protons of cytidine. This discrepancy suggests that the conformation of this residue is different in the mononucleotides relative to double helices.     

Selective catalysis of A.T base pair proton exchange in DNA complexes: imino proton NMR analysis.

Polyaromatic molecules with amino chain substituents, upon binding with DNA, selectively catalyze exchange of the A.T base pair protons with bulk water protons. The amine-catalyzed exchange is mediated by compounds which are A.T and G.C base sequence specific, intercalators, and outside binders. A mechanism for the selective exchange, involving transient opening and closing of individual A.T base pairs in the duplex, is discussed.     

Equilibrium between a wobble and ionized base pair formed between fluorouracil and guanine in DNA as studied by proton and fluorine NMR

A synthetic oligonucleotide duplex containing the chemotherapeutic and mutagenic agent 5-fluorouracil paired with guanine has been studied in solution by proton and fluorine NMR. The 7-mer duplex containing a central FU.G base pair adopts a normal right-handed configuration. At low pH, the predominant base-paired structure is wobble, whereas at higher pH an ionized structure in Watson-Crick geometry is observed. The two structures are in a pH-dependent equilibrium with one another with an apparent pK of 8.3 at 23 degrees C. This is the first demonstration of an equilibrium between two distinct base pairing schemes and the first demonstration of a negatively charged base pair in DNA.     

Chemical evidence that chromatin DNA exists as 160 base pair beads interspersed with 40 base pair bridges.

Digestion of rat liver nuclei by an endogenous endonuclease generates double-stranded DNA fragments which are initially about 205 base pairs long, as reported previously by Hewish and Burgoyne. As digestion proceeds, the average size of these fragments is reduced to about 160 base pairs. Electrophoresis under denaturing conditions shows that these DNA fragments contain single strand nicks at ten base intervals. Fifteen bands, 10-150 bases, are clearly resolvable. DNA Fragments of 160 to 200 nucleotides are not resolved as distinct species. The results suggest that the chromosomal subunit contains both a 160 base-pair DNA segment, in a conformation susceptible to single strand nicking at ten base intervals, and a forty base-pair DNA segment in a conformation more uniformly susceptible to endogenous endonuclease activity. This chemical evidence agrees with morphological observations suggesting that chromatin has a "bead and bridge" structure.     

Highly specific unnatural base pair systems as a third base pair for PCR amplification

Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ～10(10)-fold by 40 cycles of PCR, and the selectivity of the Ds-Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds-Px pair in DNA survived in the 10(28)-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds-Px pair system provides a framework for new biotechnology.     

Random coil proton chemical shifts of deoxyribonucleic acids

Sixteen 17-nucleotide DNA sequences have been used to determine the sequence effect on random coil DNA proton chemical shifts. Based on the proton chemical shifts measured for the central nucleotides in 64 triplets and the correction factors determined for the next nearest neighbor effects, a parameter set has been derived for predicting random coil DNA proton chemical shifts. The root-mean-square deviation (RMSD) between the predicted and the observed aromatic H6/H8 proton chemical shifts of 200 data from 22 random coil DNA sequences was determined to be 0.02 ppm with a correlation coefficient of 0.998. For the H1, H2, H2 and H3 sugar protons, the RMSD values between the predicted and the experimental shifts were found to be 0.02, 0.03, 0.03 and 0.02 ppm, respectively.     

2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases.

DNA base flipping, which was first observed for the C5-cytosine DNA methyltransferase M. Hha I, results in a complete removal of the stacking interactions between the target base and its neighbouring bases. We have investigated whether duplex oligodeoxynucleotides containing the fluorescent base analogue 2-aminopurine can be used to sense DNA base flipping. Using M. Hha I as a paradigm for a base flipping enzyme, we find that the fluorescence intensity of duplex oligodeoxynucleotides containing 2-aminopurine at the target site is dramatically enhanced (54-fold) in the presence of M. Hha I. Duplex oligodeoxynucleotides containing 2-aminopurine adjacent to the target cytosine show little fluorescence increase upon addition of M. Hha I. These results clearly demonstrate that duplex oligodeoxynucleotides containing 2-aminopurine at the target site can serve as fluorescence probes for base flipping. Another enzyme hypothesized to use a base flipping mechanism is the N6-adenine DNA methyltransferase M. Taq I. Addition of M. Taq I to duplex oligodeoxynucleotides bearing 2-aminopurine at the target position, also results in a strongly enhanced fluorescence (13-fold), whereas addition to duplex oligodeoxynucleotides containing 2-aminopurine at the 3'- or 5'-neighbouring position leads only to small fluorescence increases. These results give the first experimental evidence that the adenine-specific DNA methyltransferase M. Taq I also flips its target base.     

Solution structure of a DNA duplex containing a formamide-adenine base pair

The N-(2-deoxy-beta3-D-erythro-pentofuranosyl) formamide residue results from a ring fragmentation product of thymine or cytosine. The presence of a formamide-adenine base pair in the sequence 5'd(AGGAACCACG).d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. There are two possible isomers for the formamide side chain, either cis or trans. For each isomer, we observed an equilibrium in solution between two forms. First, a species where the formamide is intrahelical and paired with the facing adenine. For the cis isomer, the formamide is in a syn conformation and two hydrogen bonds with adenine are formed. The trans isomer is in an anti conformation and a single hydrogen bond is observed. In the second form, whatever the isomer, the formamide is rejected outside the helix, whereas the adenine remains inside.     

Changes in C NMR chemical shifts of DNA as a tool for monitoring drug interactions

The antibiotic drug, netropsin, was complexed with the DNA oligonucleotide duplex [d(GGTATACC)]₂ to explore the effects of ligand binding on the ¹³C NMR chemical shifts of the DNA base and sugar carbons. The binding mode of netrospin to TA-rich tracts of DNA has been well documented and served as an attractive model system. For the base carbons, four large changes in resonance chemical shifts were observed upon complex formation: −0.64 ppm for carbon 4 of either Ado4 or Ado6, 1.36 ppm for carbon 2 of Thd5, 1.33 ppm for carbon 5 of Thd5 and 0.94 for carbon 6 of Thd5. AdoC4 is covalently bonded to a heteroatom that is hydrogen bonded to netropsin; this relatively large deshielding is consistent with the known hydrogen bond formed at AdoN3. The three large shielding increases are consistent with hydrogen bonds to water in the minor groove being disrupted upon netropsin binding. For the DNA sugar resonances, large changes in chemical shifts were observed upon netropsin complexation. The 2′, 3′ and 5′ ¹³C resonances of Thd3 and Thd5 were shielded whereas those of Ado4 and Ado6 were deshielded; the ¹³C resonances of 1′ and 4′ could not be assigned. These changes are consistent with alteration of the dynamic pseudorotational states occupied by the DNA sugars. A significant alteration in the pseudorotational states of Ado4 or Ado6 must occur as suggested by the large change in chemical shift of −1.65 ppm of the C3′ carbon. In conclusion, ¹³C NMR may serve as a practical tool for analyzing structural changes in DNA-ligand complexes.     

Sequence-dependent base pair opening in DNA double helix

Preservation of genetic information in DNA relies on shielding the nucleobases from damage within the double helix. Thermal fluctuations lead to infrequent events of the Watson-Crick basepair opening, or DNA "breathing", thus making normally buried groups available for modification and interaction with proteins. Fluctuational basepair opening implies the disruption of hydrogen bonds between the complementary bases and flipping of the base out of the helical stack. Prediction of sequence-dependent basepair opening probabilities in DNA is based on separation of the two major contributions to the stability of the double helix: lateral pairing between the complementary bases and stacking of the pairs along the helical axis. The partition function calculates the basepair opening probability at every position based on the loss of two stacking interactions and one base-pairing. Our model also includes a term accounting for the unfavorable positioning of the exposed base, which proceeds through a formation of a highly constrained small loop, or a ring. Quantitatively, the ring factor is found as an adjustable parameter from the comparison of the theoretical basepair opening probabilities and the experimental data on short DNA duplexes measured by NMR spectroscopy. We find that these thermodynamic parameters suggest nonobvious sequence dependent basepair opening probabilities.     

Proton exchange and base pair opening in a DNA triple helix

Nuclear magnetic resonance spectroscopy has been used to characterize opening reactions and stabilities of individual base pairs in two related DNA structures. The first is the triplex structure formed by the DNA 31-mer 5'-AGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3'. The structure belongs to the YRY (or parallel) family of triple helices. The second structure is the hairpin double helix formed by the DNA 20-mer 5'-AGAGAGAACCCCTTCTCTCT-3' and corresponds to the duplex part of the YRY triplex. The rates of exchange of imino protons with solvent in the two structures have been measured by magnetization transfer from water and by real-time exchange at 10 degrees C in 100 mM NaCl and 5 mM MgCl2 at pH 5.5 and in the presence of two exchange catalysts. The results indicate that the exchange of imino protons in protonated cytosines is most likely limited by the opening of Hoogsteen C+G base pairs. The base pair opening parameters estimated from imino proton exchange rates suggest that the stability of individual Hoogsteen base pairs in the DNA triplex is comparable to that of Watson-Crick base pairs in double-helical DNA. In the triplex structure, the exchange rates of imino protons in Watson-Crick base pairs are up to 5000-fold lower than those in double-helical DNA. This result suggests that formation of the triplex structure enhances the stability of Watson-Crick base pairs by up to 5 kcal/mol. This stabilization depends on the specific location of each triad in the triplex structure.     

Triple helical DNA in a duplex context and base pair opening

It is fundamental to explore in atomic detail the behavior of DNA triple helices as a means to understand the role they might play in vivo and to better engineer their use in genetic technologies, such as antigene therapy. To this aim we have performed atomistic simulations of a purine-rich antiparallel triple helix stretch of 10 base triplets flanked by canonical Watson–Crick double helices. At the same time we have explored the thermodynamic behavior of a flipping Watson–Crick base pair in the context of the triple and double helix. The third strand can be accommodated in a B-like duplex conformation. Upon binding, the double helix changes shape, and becomes more rigid. The triple-helical region increases its major groove width mainly by oversliding in the negative direction. The resulting conformations are somewhere between the A and B conformations with base pairs remaining almost perpendicular to the helical axis. The neighboring duplex regions maintain a B DNA conformation. Base pair opening in the duplex regions is more probable than in the triplex and binding of the Hoogsteen strand does not influence base pair breathing in the neighboring duplex region.     

Photoinduced double proton transfer in a model hydrogen bonded base pair. Theoretical study

A theoretical study of the 7-azaindole dimer confirms the photoinduced biprotonic transfer mechanism in the first ($\text{π, π}{}^1$) excited singlet state. Emission is forbidden from the first excited singlet state (A_g) of the normal form and it is allowed from the same state (B_u) of a tautomeric form. Proton transfer depends very strongly on the distance between the two monomer molecules, and extension of this mechanism to explain U.V. induced mutation in DNA is conditioned to the possibility of its basis attaining the optimal separation.     

Phosphorus-31 nuclear magnetic resonance of double- and triple-helical nucleic acids. Phosphorus-31 chemical shifts as a probe of phosphorus-oxygen ester bond torsional angles

The temperature dependence to the 31P NMR spectra of poly[d(GC)] . poly [d(GC)],d(GC)4, phenylalanine tRNA (yeast) and mixtures of poly(A) + oligo(U) is presented. The 31P NMR spectra of mixtures of complementary RNA and of the poly d(GC) self-complementary DNA provide torsional information on the phosphate ester conformation in the double, triple, and "Z" helix. The increasing downfield shift with temperature of the single-strand nucleic acids provides a measure of the change in the phosphate ester conformation in the single helix to coil conversion. A separate upfield peak (20-60% of the total phosphates) is observed at lower temperatures in the oligo(U) . poly(A) mixtures which is assigned to the double helix/triple helix. Proton NMR and UV spectra confirm the presence of the multistrand forms. The 31P chemical shift for the double helix/triple helix is 0.2-0.5 ppm upfield from the chemical shift for the single helix which in turn is 1.0 ppm upfield from the chemical shift for the random coil conformation.     

Analysis of proton chemical shifts in regular secondary structure of proteins

Summary The contribution of peptide groups to H and H proton chemical shifts can be modeled with empirical equations that represent magnetic anisotropy and electrostatic interactions [Ösapay, K. and Case, D.A. (1991) J. Am. Chem. Soc., 113, 9436–9444]. Using these, a model for the random coil reference state can be generated by averaging a dipeptide over energetically allowed regions of torsion-angle space. Such calculations support the notion that the empirical constant used in earlier studies arises from neighboring peptide contributions in the reference state, and suggest that special values be used for glycine and proline residues, which differ significantly from other residues in their allowed ,-ranges. New constants for these residues are reported that provide significant improvements in predicted backbone shifts. To illustrate how secondary structure affects backbone chemical shifts we report calculations on oligopeptide models for helices, sheets and turns. In addition to suggesting a physical mechanism for the widely recognized average difference between and secondary structures, these models suggest several additional regularities that should be expected: (a) H protons at the edges of -sheets will have a two-residue periodicity; (b) the H² and H³ protons of glycine residues will exhibit different shifts, particularly in sheets; (c) H protons will also be sensitive to local secondary structure, but in different directions and to a smaller extent than H protons; (d) H protons in turns will generally be shifted upfield, except those in position 3 of type I turns. Examples of observed shift patterns in several proteins illustrate the application of these ideas.