Flexibility of A.T pairs in left-handed DNA helices

We have analysed by various approaches the structure of cloned synthetic sequences in supercoiled plasmids. Individual inserts were formed by d(C-G)n blocks interrupted by the presence of A.T pairs positioned either in phase or out of phase of pur-pyr alternation. Based on the thermodynamic analysis we obtained results confirming that A.T pairs are easily incorporated into left-handed helices without significant energetic penalty. Sequences GTAC which are known to form cruciform structures in multiple repetition underwent a B-Z transition. In the case of plasmids containing AA/TT code words and substantial discontinuities in purine-pyrimidine alternation our analysis indicates that Z-Z junctions formed by A.T pairs contributed little to the overall energetic demands of the B-Z transition probably thanks to their high conformational flexibility.     

Synthesis and monitored selection of nucleotide surrogates for binding T:A base pairs in homopurine-homopyrimidine DNA triple helices

A total of 16 oligodeoxyribonucleotides of general sequence 5′-TCTTCTZTCTTTCT-3′, where Z denotes an N-acyl-N-(2-hydroxyethyl)glycine residue, were prepared via solid phase synthesis. The ability of these oligonucleotides to form triplexes with the duplex 5′-AGAAGATAGAAAGA-HEG-TCTTTCTATCTTCT-3′, where HEG is a hexaethylene glycol linker, was tested. In these triplexes, an ‘interrupting’ T:A base pair faces the Z residue in the third strand. Among the acyl moieties of Z tested, an anthraquinone carboxylic acid residue linked via a glycinyl group gave the most stable triplex, whose UV melting point was 8.4°C higher than that of the triplex with 5′-TCTTCTGTCTTTCT-3′ as the third strand. The results from exploratory nuclease selection experiments suggest that a combinatorial search for strands capable of recognizing mixed sequences by triple helix formation is feasible.     

Sequence effects on the propeller twist of base pairs in DNA helices.

Molecular mechanics calculations are performed on all the ten base pair steps (duplex dimers) and also a number of trimer and tetramer duplexes comprising them, in an attempt to systematically examine the possible base sequence effects on the magnitudes of propeller twists of base pairs at a given step. The analysis reveals that though propeller twist is a base pair property, it behaves very much like other base step parameters such as slide, roll, twist etc., Hence, it may be necessary to monitor the nature and variation of magnitudes of pt at a step. Calculations performed on 45 out of the 136 unique tetramer combinations involving all the ten unique base steps show that the difference in magnitudes of propeller twists of the base pairs of a given step has been found to be either steep or moderate depending on base pairs that flank the base step. These observations compare very well with the available experimental data. Tetramer sequences, wherein a base pair of a base step repeats in the same direction, exhibit a relatively steep difference in propeller twist at the step. Tetramers other than these exhibit moderate difference in propeller twist. Such sequences are broadly classified as type-I and type-II respectively. Practically all the tetrads considered in the study, excepting those with GT step and a few involving CG and GC steps, conform to the above classification.     

Effects of A:T base pairs on the B-Z conformational transitions of DNA

Effects of A:T base pairs on the propensity of B to Z conformational transitions have been investigated by the CD salt titrations on d(CG)5' d(GC)5' terminal or central A:T replaced decamers, and terminal A:T appended dodecamers. The presence of A:T at the center greatly inhibits the B to Z transition of both G:C decamers. Moderate Z inhibitions are shown by terminal A:T replacements and additions to d(CG)5' with the former exhibiting a stronger effect. In contrast, the addition and replacement with A:T at the terminals of d(GC)5 facilitate the B to Z conversion, with the replacement exhibiting a somewhat more pronounced effect. These results may be rationalized in terms of the number of contigous CG sequences present in an oligomer and the relative inhibitory effects of other dinucleotide sequences. Our results also suggest that some short oligomers with purine at the 5'-end, such as d[A(CG)nT] with n greater than or equal to 2, may likely crystallize as Z conformations.     

Trans-hydrogen bond deuterium isotope effects of A:T base pairs in DNA

The chemical shifts of (13)C2 of adenosine residues of DNA were observed to experience a through-space or trans-hydrogen bond isotope effect as a result of deuterium substitution at the imino hydrogen site of base-paired thymidine residues. NMR measurements of several self-complementary DNA duplexes at natural abundance (13)C in 50% H(2)O, 50% D(2)O solvent mixtures yielded an average trans-hydrogen bond isotope effect, (2h)Delta(13)C2, of -47 ppb. The data suggest that stronger hydrogen bonds have more negative (2h)Delta(13)C2 values, which means that A:T N1.H3 hydrogen bonds increase the anharmonicity of the effective vibrational potential of H3. However, (2h)Delta(13)C2 values do not correlate with intra-residue (2)Delta(13)C4 values of thymidine observed here and earlier (Vakonakis et al., 2003), which suggests that (2h)Delta(13)C2 is not determined entirely by hydrogen bond strength. Instead, the variations observed in (2h)Delta(13)C2 values suggest that they may also be sensitive to base pair geometry.     

Recognition of base pairs by polar peptides in double stranded DNA.

The resonance Raman spectrum of native DNA has been obtained using excitation at 257 nm. In a first part, the spectral lines are assigned to the different nucleotide bases which provide the resonance effect. In a second part, the interactions of DNA with basic peptides (Arginine Methylester, Lysine Methylester, Arginyl-Arginine) are investigated using excitation at 300 nm and 257 nm, which give complementary information about the DNA. Both Arginine Methylester and Arginyl-arginine recognize the A-T base pairs, the first one in the large groove, the second one in the narrow groove of DNA. The DNA-Lysine Methylester interaction is very likely not specific but can take place in the large groove of DNA.     

Vibrational fluctuations of hydrogen bonds in a DNA double helix with nonuniform base pairs.

The Green's function technique is applied to a study of breathing modes in a DNA double helix which contains a region of different base pairs from the rest of the double helix. The calculation is performed on a G-C helix in the B conformation with four consecutive base pairs replaced by A-T. The average stretch in hydrogen bonds is found amplified around the A-T base pair region compared with that of poly(dG)-poly(dC). This is likely related to the A-T regions lower stability against hydrogen bond melting. The A-T region may be considered to be the initiation site for melting in such a helix.     

Deuterium isotope effects and fractionation factors of hydrogen-bonded A:T base pairs of DNA

Deuterium isotope effects and fractionation factors of N1...H3–N3 hydrogen bonded Watson–Crick A:T base pairs of two DNA dodecamers are presented here. Specifically, two-bond deuterium isotope effects on the chemical shifts of ¹³C2 and ¹³C4, ²¹³C2 and ²¹³C4, and equilibrium deuterium/protium fractionation factors of H3, , were measured and seen to correlate with the chemical shift of the corresponding imino proton, _H3. Downfield-shifted imino protons associated with larger values of ²¹³C2 and ²¹³C4 and smaller values, which together suggested that the effective H3–N3 vibrational potentials were more anharmonic in the stronger hydrogen bonds of these DNA molecules. We anticipate that ²¹³C2, ²¹³C4 and values can be useful gauges of hydrogen bond strength of A:T base pairs.     

A new model for DNA containing A.T and I.C base pairs.

DNA polymers containing exclusively A.T or I.C base pairs frequently exhibit D- or E-type X-ray diffraction patterns when dried. The distribution of intensities in fiber patterns appears to demand helical structures with 7 and 7.5 bp/turn, respectively, but it is not stereochemically possible to wind a right-handed antiparallel B-family helix this tightly. It is a simple matter, however, to build a left-handed helix with 7-7.5 bp/turn by incorporating Hoogsteen pairing into a Z helix framework. X-ray intensities calculated from this novel left-handed Hoogsteen model provide as reasonable a fit to the D-DNA diffraction pattern as do intensities calculated from previously proposed right-handed 8-fold models.     

Tracts of A.T base pairs retard the electrophoretic mobility of short DNA duplexes.

An investigation of the mobility of short duplexes of DNA -octamers and decamers-on polyacrylamide gels is presented, showing that molecules containing less than one helical turn exhibit sequence dependent mobilities. Analysis of chains with different sequences indicates that any arrangement of two or more adjacent A.T base pairs causes a duplex to move more slowly than does any combination of isolated A.T pairs This behavior appears to be an intrinsic property of these sequences, since the anomaly persists in the absence of magnesium or presence of spermine and is not due to strand dissociation. In two decamers we studied, the position of A.T tracts within a duplex can be shown to influence mobility: the sequence GA4T4C associated with bending or curvature of the helix axis when ligated into polymers migrates more slowly than the corresponding sequence GT4A4C, polymers of which migrate as linear B DNA.     

Dynamics of the B-A transition of DNA double helices

Although the transition from the B-DNA double helix to the A-form is essential for biological function, as shown by the existence of the A-form in many protein–DNA complexes, the dynamics of this transition has not been resolved yet. According to molecular dynamics simulations the transition is expected in the time range of a few nanoseconds. The B–A transition induced by mixing of DNA samples with ethanol in stopped flow experiments is complete within the deadtime, showing that the reaction is faster than ~0.2 ms. The reaction was resolved by an electric field jump technique with induction of the transition by a dipole stretching force driving the A- to the B-form. Poly[d(A-T)] was established as a favourable model system, because of a particularly high cooperativity of the transition and because of a spectral signature allowing separation of potential side reactions. The time constants observed in the case of poly[d(A-T)] with ~1600 bp are in the range around 10 µs. An additional process with time constants of ~100 µs is probably due to nucleation. The same time constants (within experimental accuracy ±10%) were observed for a poly[d(A-T)] sample with ~70 bp. Under low salt conditions commonly used for studies of the B–A transition, the time constants are almost independent of the ionic strength. The experimental data show that a significant activation barrier exists in the B–A transition and that the helical states are clearly separated from each other, in contrast to predictions by molecular dynamics simulations.     

Excited states of the DNA base pairs

The results of calculations of the first π-electronic states of the DNA base pairs with the SCF-MO-LCAO method both without taking into account configuration interaction and taking into account all the singly excited configurations are presented. The first excited singlet state and the first triplet state of both pairs are shown to be, independently of an approximation, the states where the excitation is localized on one of the bases.     

Parallel double helices of DNA. Conformational analysis of regular helices with the second order symmetry axis

We have performed a conformational analysis of DNA double helices with parallel directed backbone strands connected with the second order symmetry axis being at the same time the helix axis. The calculations were made for homopolymers poly(dA).poly(dA), poly(dC).poly(dC), poly(dG) poly(dG), and poly(dT).poly(dT). All possible variants of hydrogen bonding of base pairs of the same name were studied for each polymer. The maps of backbone chain geometrical existence were constructed. Conformational and helical parameters corresponding to local minima of conformational energy of "parallel" DNA helices, calculated at atom-atom approximation, were determined. The dependence of conformational energy on the base pair and on the hydrogen bond type was analysed. Two major conformational advantageous for "parallel" DNA's do not depend much on the hydrogen-bonded base pair type were indicated. One of them coincided with the conformational region typical for "antiparallel" DNA, in particular for the B-form DNA. Conformational energy of "parallel" DNA depends on the base pair type and for the most part is similar to the conformational energy of "antiparallel" B-DNA.     

Spin-orbit couplings in the DNA base pairs

The radiative lifetimes of the phosphorescent states of the adenine.thymine (A.T) and guanine.cytosine (G.C) base pairs were calculated on the basis of the singlet-triplet transition probability induced by spin-orbit couplings. The calculated radiative lifetimes averaged over the triplet sublevels of spin state were in the order of G.C less than A.T and in good correlation with those of the composite bases. On the whole the results suggested an important role for thymine triplet having a relatively long lifetime during the course of the triplet localization in DNA, in agreement with the experimental observation that the concentration of triplet is remarkably enhanced with increase in A+T content.     

Circular dichroism spectra of DNA oligomers show that short interior stretches of C.C+ base pairs do not form in duplexes with A.T base pairs

Circular dichroism (CD) experiments were carried out on a series of DNA oligomers to determine if short internal stretches of protonated cytosine-cytosine (C.C+) base pairs could coexist with adenine-thymine (A.T) base pairs. (1) C.C+ base pairs did form in the absence of A.T base pairs in the individual oligomers d(AACC)5 and d(CCTT)5, as indicated by the appearance of a long-wavelength CD band centered at 282-284 nm, when the pH was lowered to 6 or 5 at 0.5 M Na+. A comparison of measured with calculated spectra showed that d(CCTT)5 at pH 5, 0.5 M Na+, 20 degrees C, likely adopted a structure with a central core of stacked C.C+ base pairs and looped-out thymines. Under the same conditions, it appeared that C.C+ base pairs also formed in d(AACC)5, but with the adenines remaining intrahelical. Each of these oligomers showed a cooperative transition for formation of C.C+ base pairs as the temperature was lowered, with C.C+ base pairs forming at a higher temperature in d(CCTT)5 than in d(AACC)5. A.T base formed in equimolar mixtures of d(AACC)5 plus d(CCTT)5 as monitored by an increase in the negative magnitude of the 250-nm CD band. However, a large increase did not appear at about 285 nm in CD spectra of the mixtures, showing that there were no stacked C.C+ base pairs in the d(AACC)5.d(CCTT)5 duplex even though they formed under the same conditions in the individual strands. Thus, in this duplex, A.T base pairs prevented the formation of neighboring internal C.C+ base pairs. (2) CD measurements were also made of d(A10C4T10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of mismatched base pairs in DNA

The structural dynamics of mismatched base pairs in duplex DNA have been studied by time-resolved fluorescence anisotropy decay measurements on a series of duplex oligodeoxynucleotides of the general type d[CGG(AP)GGC].d[GCCXCCG], where AP is the fluorescent adenine analogue 2-aminopurine and X = T, A, G, or C. The anisotropy decay is caused by internal rotations of AP within the duplex, which occur on the picosecond time scale, and by overall rotational diffusion of the duplex. The correlation time and angular range of internal rotation of AP vary among the series of AP.X mismatches, showing that the native DNA bases differ in their ability to influence the motion of AP. These differences are correlated with the strength of base-pairing interactions in the various AP.X mismatches. The interactions are strongest with X = T or C. The ability to discern differences in the strength of base-pairing interactions at a specific site in DNA by observing their effect on the dynamics of base motion is a novel aspect of the present study. The extent of AP stacking within the duplex is also determined in this study since it influences the excited-state quenching of AP. AP is thus shown to be extrahelical in the AP.G mismatch. The association state of the AP-containing oligodeoxynucleotide strand is determined from the temperature-dependent tumbling correlation time. An oligodeoxynucleotide triplex is formed with a particular base sequence in a pH-dependent manner.     

The mechanism of ion polarisation along DNA double helices

The orientation curves of short DNA fragments induced by electric field pulses are measured with high time resolution and analysed by efficient deconvolution techniques. A small, but clearly detectable delay of the 'on-field' orientation can be described accurately by the superposition of two exponential processes with opposite amplitudes. The time constant of the faster process is around 10 ns and the slower one in the range 50-1000 ns depending upon the electric field strength and chain length of the DNA fragment. The relation between amplitudes and time constants observed for each curve corresponds exactly to that expected for a convolution of two processes, where the first process is without optical response and becomes detectable only via the optical response of the second process. These results indicate that the first process reflects the polarisation of the ion atmosphere required for the second process of the orientation. Measurements at different ion concentrations c demonstrate that the reciprocal time constant of the fast process is a linear function of c and thus is consistent with an association reaction. The association rate constant evaluated from this dependence according to a simple bimolecular reaction model is 8 X 10(9) M-1 s-1 for a 95 base-pair fragment and is consistent with binding of Na+ to the helix, a reaction close to the limit of diffusion control. The association rate constant is almost independent of the electric field strength E, while the dissociation rate constant k- strongly increases with E, indicating dissociation of ions at high E values. The data suggest a linear correlation between log(k-) and E2 corresponding to a reaction driven by a dipole change. The apparent dipole change evaluated from this dependence is in the order of magnitude estimated for an elementary step of ion dissociation at one end of the helices. The combined results obtained from the polarisation and the orientation mechanism can be explained by dissociation of surprisingly few counterions biased towards one end of the helices. The experimental data obtained for a 76 base-pair fragment are analogous to those for the 95 base-pair fragment, whereas the 'slow' ion polarisation has not been detected for a fragment with 27 base-pairs. This result together with those obtained for the longer fragments at low field strengths indicate that there is a fast polarisation mechanism without 'ion dissociation' at low chain lengths and for low electric field strengths. This mechanism is replaced at high chain lengths and/or high electric field strengths by the ion dissociation mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alternative heterocycles for DNA recognition: an N-methylpyrazole/N-methylpyrrole pair specifies for A.T/T.A base pairs

Side-by-side pairs of three five-membered rings, N-methylpyrrole (Py), N-methylimidazole (Im), and N-methylhydroxy-pyrrole (Hp), have been demonstrated to distinguish each of the four Watson Crick base pairs in the minor groove of DNA. However, not all DNA sequences targeted by these pairing rules achieve affinities and specificities comparable to DNA binding proteins. We have initiated a search for new heterocycles which can expand the sequence repetoire currently available. Two heterocyclic aromatic amino acids. N-methylpyrazole (Pz) and 4-methylthiazole (Th), were incorporated into a single position of an eight-ring polyamide of sequence ImImXPy-gamma-lmPyPyPy-beta-Dp to examine the modulation of affinity and specificity for DNA binding by a Pz/Py pair and or a Th/Py pair. The X/Py pairings Pz/Py and Th/Py were evaluated by quantitative DNase I footprint titrations on a DNA fragment with the four sites 5'-TGGNCA-3' (N=T, A, G, C). The Pz/Py pair binds T.A and A.T with similar affinity to a Py/Py pair but with improved specificity. disfavoring both G.C and C.G by about 100-fold. The Th/Py pair binds poorly to all four Watson Crick base pairs. These results demonstrate that in some instances new heterocyclic aromatic amino acid pairs can be incorporated into imidazole-pyrrole polyamides to mimic the DNA specificity of Py/Py pairs which may be relevant as biological criteria in animal studies become important.     

Electrochemical DNA base pairs quantification and endonuclease cleavage detection

A label-free multiplexed immunoassay strategy was proposed for the simultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Monoclonal antibody of CEA was co-immobilized with ferrocenecarboxylic acid (FCA) inside the channels of mesoporous silica (MPS) to prepare the label-free probe for CEA. Also, monoclonal antibody of AFP was co-immobilized with horseradish peroxidase (HRP) inside the channels of MPS to prepare the label-free probe for AFP by using o-phenylenediamine (OPD) and H(2)O(2) as the electrochemical substrates. Thus, the multianalyte immunosensor was constructed by coating the probes of CEA and AFP respectively onto the different areas of indium-tin oxide (ITO) electrode. When the immunosensor was incubated with sample antigens, CEA and AFP antigens were introduced into the mesopores of MPS after the immunoassay reaction. Because all of the Si-OH groups on the external surface of MPS were blocked with Si(CH(3))(3), the proteins and substrates were limited to be embedded on the internal pore walls. Therefore, the electric response transfer was confined inside the pore channels. The nonconductive immunoconjugates blocked the electron transfer and the peak responses changed on the corresponding surface respectively. Then, the simultaneous detection of CEA and AFP achieved. The linear ranges of CEA and AFP were 0.5-45ngmL(-1) and 1-90ngmL(-1) with the detection limits of 0.2ngmL(-1) and 0.5ngmL(-1) (S/N=3), respectively. The fabricated immunosensor shows appropriate sensitivity and offers an alternative to the multianalyte detection of antigens or other bioactive molecules.