Raman spectra of L-alanine oligomers

The Raman spectra have been obtained of di-, tri-, tetra-, penta-, and hexa-L -alanine in the solid state. Raman spectra of the dimer and trimer in aqueous solution are also reported. The oligomers of alanine exist as zwitterions in the solid state and aqueous solution. Spectral differences between the dipeptide, and other oligomers arise primarily from the conformationally sensitive amide modes. The dipeptide exists as a nonplanar structure in the solid state and the other oligomere as β conformations. Comparison of Raman spectra of tri-L -alanine in the solid state and in aqueous solution suggests a conformational change to a random coil upon dissolution.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

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)     

Numerical simulations of Raman spectra of guanine-cytosine Watson-Crick and protonated Hoogsteen base pairs

Large changes in the Raman spectra of calf thymus DNA are observed upon lowering the pH. In order to gain a better insight into these effects, several simulations of the Raman spectra of the guanine-cytosine (GC) Watson-Crick and Hoogsteen base pairs are performed. By comparing the Raman bands of GC base pairs in calf thymus DNA at high and low pH with the theoretical simulations of GC base pairs, it is found that the intensity changes in the theoretical bands located between 400 and 1000 cm(-1) are small compared to the experimental ones. The behavior of the cytosine band at 1257 cm(-1) upon lowering the pH is not reproduced in the GC theoretical spectra. The bands located above 1300 cm(-1) in the theoretical spectra display intensity changes that are similar to those found for GC base pairs in calf thymus DNA spectra.     

Genetic and quantum chemical basis of the mutagenicity of nitroarenes for adenine-thymine base pairs

The mutagenicity of nitroarenes for Salmonella typhimurium strains with adenine-thymine base pairs at the mutational site is dependent upon enzymic reduction of the nitro function. Although the electrophilic metabolites of nitroarenes are capable of mutating adenine-thymine base pairs, they show a marked preference for guanine-cytosine pairs when given a choice. Quantum chemical calculations indicate the reactivity order for nucleophilic sites in an AT run of base pairs to be the N-7 of adenine (N7(A)) first, followed by an approximately equal reactivity for C-8 of adenine (C8(A)) and O4 of thymine (O4(T)). Given the low probability of reaction of electrophilic metabolites of nitroarenes with adenine-thymine base pairs, the mutagenic potency of nitroarenes for strains with adenine-thymine base pairs at the mutational site is remarkable.     

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.     

DNA base modification: ionized base pairs and mutagenesis

The nature of hydrogen bonding between normal and modified bases has been re-examined. It is proposed that hydrogen-bonding schemes may involve tautomeric, ionized or conformational forms (syn, anti and wobble). Several important cases are presented or reviewed in which physical evidence indicates the existence of ionized base pairs. When thermodynamic values determined in aqueous solution under physiological conditions are considered, it can be argued that base ionization will contribute substantially to the stability of many biologically relevant base pairs containing modified bases. A significant incidence of ionized bases in DNA may have important kinetic ramifications for the further chemical reactivity of both the modified base and its cross-strand pairing partner. Moreover, DNA structure at and surrounding ionized base pairs may be altered. For this reason, the model presented in this study should be useful as DNA-sequence analysis becomes more commonly applied to the study of mutagenesis.     

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.     

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.     

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.     

Thermodynamics and kinetics of the helix-coil transition of oligomers containing GC base pairs

Absorbance-temperature profiles have been determined for the following self-complementary oligonucleotides or equimolar paris of complementary oligonucleotides containing GC base pairs: A₂GCU₂, A₃GCU₃, A₄GCU₄, A₆CG + CGU₆, A₈CG + CGU₈, A₄G₂ + C₂U₄, A₅G₂ + C₂U₅, A₄G₃ + C₃U₄, and A₅G₃ + C₃U₅. In all cases cooperative melting transitions indicate double-helix formation. As was found previously, the stability of GC containing oligomer helices is much higher than that of AU helices of corresponding length. Moreover, helices with the same length and base composition but different sequences also have quite different stabilites. The melting curves were andlyzed using a zipper model and the thermodynamic parameters for the AU pairs determined previously. The effect of single-strand stacking was considered separately. According to this model, the formation of a GC pair from unstacked single strands is associated with an ethalpy change of ?15 kcal/mole. Due to the high degree of single-strand stacking at room temperature the enthalpy change for the formation of GC pairs from unstacked single strands is only ?5 to ?6 kcal/mole. (The corresponding parameters for AU pairs are ?10.7 kcal/mole and ?5 to ?6 kcal/mole.) The sequence dependence of helix stability seems to be primarily entropic since no differences in ΔH were seen among the sequence isomers. The kinetics of helix formation was investigated for the same molecules using the temperature jump technique. Recombination of strands is second order with rate constants in the range of 10⁵ to 10⁷M^?1 sec^?1 depending on the chain length and the nucleotide sequence. Within a series of oligomers of a given type, the rates of recombination decrease with increasing chain length. Oligomers with the sequence A_nGCU_n recombine six to eight times slower than the other oligomers of corresponding chain length. The experimental enthalpies of activation of 6 to 9 kcal/mole suggest a nucleation length of one or two GC base pairs. The helix dissociation process has rate constants between 0.5 and 500 sec^?1 and enthalpies of activation of 25 to 50 kcal/mole. An increase of chain length within a given nucleotide series leads to decreased rates of dissociation and increased enthalpies of activation. An investigation of the effect of ionic strength on A_nGCU_n helix formation showed that the rates of recombination increase considerably with increased ionic strength.     

Nitrogen-15 chemical shifts in AT (adenine-thymine) and CG (cytosine-guanine) nucleic acid base pairs

This paper presents ab initio (DFT) calculations of the 15N chemical shifts in AT (Adenine-Thymine) and CG (Cytosine-Guanine) nucleic acid base pairs. Calculations were done on 14 AT and 18 CG base pairs using experimental (X-ray) geometries obtained from several DNA decamers. The calculated chemical shifts are compared with the experimental values in the pure bases and subjected to statistical analysis to explore their sensitivity to the local geometry and pair helix parameters. The results indicate that the 15N chemical shifts, isotropic and principal components are quite sensitive to small changes in the geometry of the pairs, but they do not correlate well with the helix pair parameters. From the statistical analysis, several linear correlations between structural parameters and chemical shifts emerge. These relationships may serve as a foundation to extract information on molecular structure from 15N chemical shift measurements.     

Do Hoogsteen base pairs occur in DNA?

The importance of the Watson-Crick complementary base-pairing scheme has rather overshadowed alternative types of base pairs in DNA. One of these alternative base pairings, which is known as Hoogsteen pairing, is now receiving attention. Its presence in crystals of oligonucleotides bound to some antibiotics, and its possible existence in solution (and within long DNA fragments) remains to be unambiguously estimated. However, variability in DNA conformation appears to play an important biological role, and thus we should consider the presence of Hoogsteen base pairs as an interesting factor in inducing such changes.     

Polarized Raman spectra of oriented fibers of A DNA and B DNA: anisotropic and isotropic local Raman tensors of base and backbone vibrations.

Polarized Raman spectra of oriented fibers of calf thymus DNA in the A and B conformations have been obtained by use of a Raman microscope operating in the 180 degrees back-scattering geometry. The following polarized Raman intensities in the spectral interval 200-1800 cm-1 were measured with both 514.5 and 488.0 nm laser excitations: (1) Icc, in which the incident and scattered light are polarized parallel to the DNA helical axis (c axis); (2) Ibb, in which the incident and scattered light are polarized perpendicular to c; and (3) Ibc and Icb, in which the incident and scattered light are polarized in mutually perpendicular directions. High degrees of structural homogeneity and unidirectional orientation were confirmed for both the A and B form fibers, as judged by comparison of the observed Raman markers and intensity anisotropies with measurements reported previously for oligonucleotide single crystals of known three-dimensional structures. The fiber Raman anisotropies have been combined with solution Raman depolarization ratios to evaluate the local tensors corresponding to key conformation-sensitive Raman bands of the DNA bases and sugar-phosphate backbone. The present study yields novel vibrational assignments for both A DNA and BDNA conformers and also confirms many previously proposed Raman vibrational assignments. Among the significant new findings are the demonstration of complex patterns of A form and B form indicator bands in the spectral intervals 750-900 and 1050-1100 cm-1, the identification of highly anisotropic tensors corresponding to vibrations of base, deoxyribose, and phosphate moieties, and the determination of relatively isotropic Raman tensors for the symmetrical stretching mode of phosphodioxy groups in A and B DNA. The present fiber results provide a basis for exploitation of polarized Raman spectroscopy to determine DNA helix orientation as well as to probe specific nucleotide residue orientations in nucleoproteins, viruses, and other complex biological assemblies.     

Effect of terminal base pairs on the CD spectra of oligodeoxynucleotide duplexes

Oligodeoxynucleotide duplexes having terminal C:G base pairs and recognition sequences for restriction endonucleases Bgl II and Sau 3Al were synthesized by the solid or liquid phase phosphotriester approach. Among them, the self-complementary oligonucleotides with C or CC at the 5'-end and G or GG at the 3'-end exhibited unusual CD spectra in contrast to those of the usual B-DNA type CD spectra of the oligonucleotide with G or GG at the 5'-end. The spectra are different to those of the Z-DNA, and regarded to the terminal base effect of the C:G pairs.     

Propynyl groups in duplex DNA: stability of base pairs incorporating 7-substituted 8-aza-7-deazapurines or 5-substituted pyrimidines

Oligonucleotides incorporating the 7-propynyl derivatives of 8-aza-7-deaza-2′-deoxyguanosine (3b) and 8-aza-7-deaza-2′-deoxyadenosine (4b) were synthesized and their duplex stability was compared with those containing the 5-propynyl derivatives of 2′-deoxycytidine (1) and 2′-deoxyuridine (2). For this purpose phosphoramidites of the 8-aza- 7-deazapurine (pyrazolo[3,4-d]pyrimidine) nucleosides were prepared and employed in solid-phase synthesis. All propynyl nucleosides exert a positive effect on the DNA duplex stability because of the increased polarizability of the nucleobase and the hydrophobic character of the propynyl group. The propynyl residues introduced into the 7-position of the 8-aza-7-deazapurines are generally more stabilizing than those at the 5-position of the pyrimidine bases. The duplex stabilization of the propynyl derivative 4b was higher than for the bromo nucleoside 4c. The extraordinary stability of duplexes containing the 7-propynyl derivative of 8-aza-7- deazapurin-2,6-diamine (5b) is attributed to the formation of a third hydrogen bond, which is apparently not present in the base pair of the purin-2,6-diamine 2′-deoxyribonucleoside with dT.     

Raman spectroscopic study of left-handed Z-RNA

The solvent conditions that induce the formation of a left-handed Z form of poly[r(G-C)] have been extended to include 6.5 M NaBr at 35 degrees C and 3.8 M MgCl2 at room temperature. The analysis of the A----Z transition in RNA by circular dichroism (CD), 1H and 31P NMR, and Raman spectroscopy shows that two distinct forms of left-handed RNA exist. The ZR-RNA structure forms in high concentrations of NaBr and NaClO4 and exhibits a unique CD signature. ZD-RNA is found in concentrated MgCl2 and has a CD signature similar to the Z form of poly[d(G-C)]. The loss of Raman intensity of the 813-cm-1 A-form marker band in both the A----ZR-RNA and A----ZD-RNA transitions parallels the loss of intensity at 835 cm-1 in the B----Z transition of DNA. A guanine vibration that is sensitive to the glycosyl torsion angle shifts from 671 cm-1 in A-RNA to 641 cm-1 in both ZD- and ZR-RNA, similar to the B----Z transition in DNA in which this band shifts from 682 to 625 cm-1. Significant differences in the glycosyl angle and sugar pucker between Z-DNA and Z-RNA are suggested by the 16-cm-1 difference in the position of this band. The Raman evidence for structural difference between ZD- and ZR-RNA comes from two groups of bands: First, Raman intensities between 1180 and 1600 cm-1 of ZD-RNA differ from those for ZR-RNA, corroborating the CD evidence for differences in base-stacking geometry. Second, the phosphodiester stretching bands near 815 cm-1 provide evidence of differences in backbone geometry between ZD- and ZR-RNA.     

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.