Synthesis, structure and thermodynamic properties of 8-methylguanine-containing oligonucleotides: Z-DNA under physiological salt conditions.

Various oligonucleotides containing 8-methylguanine (m8G) have been synthesized and their structures and thermodynamic properties investigated. Introduction Of M8G into DNA sequences markedly stabilizes the Z conformation under low salt conditions. The hexamer d(CGC[M8G]CG)2 exhibits a CD spectrum characteristic of the Z conformation under physiological salt conditions. The NOE-restrained refinement unequivocally demonstrated that d(CGC[m8G]CG)2 adopts a Z structure with all guanines in the syn conformation. The refined NMR structure is very similar to the Z form crystal structure of d(CGCGCG)2, with a root mean square deviation of 0.6 between the two structures. The contribution of m8G to the stabilization of Z-DNA has been estimated from the mid-point NaCl concentrations for the B-Z transition of various m8G-containing oligomers. The presence of m8G in d(CGC[m8G]CG)2 stabilizes the Z conformation by at least deltaG = -0.8 kcal/mol relative to the unmodified hexamer. The Z conformation was further stabilized by increasing the number of m8Gs incorporated and destabilized by incorporating syn-A or syn-T, found respectively in the (A,T)-containing alternating and non-alternating pyrimidine-purine sequences. The results suggest that the chemically less reactive m8G base is a useful agent for studying molecular interactions of Z-DNA or other DNA structures that incorporate syn-G conformation.     

Sequence dependent conformations of oligomeric DNA's in aqueous solutions and in crystals

In order to determine the sequence dependence of the conformation of deoxynucleotides, Raman spectra have been obtained for the following oligodeoxynucleotides in aqueous salt solutions and in crystals: d(CpG)(I), d(TGCGCGCA)(II), d(CACGCGTG)(III), d(CGTGCACG)(IV), d(CGCATGCG)(V), d(ACGCGCGT)(VI), d(CGCGTACGCG)(VII), d(CGCACGTGCG)(VIII) and d(CGTGCGCACG)(IX), d(GCTATAGC) (X), d(GCATATGC) (XI), d(GGTATACC) (XII) and d(GGATATCC) (XIII). The normal B type conformation is observed for all the oligomer DNA's at low salt (0.1-1.0 M NaCl) concentration in the temperature range of 0-25 degrees C. It was considered possible that all of the first nine oligomers could go into the Z form in aqueous high salt (5.0-6.0 M NaCl) solutions, and under these conditions the last four were considered candidates to go into the A form. The B-type conformation was found to exist in high salt solutions for (I), (IV), (V), (VI), (X), (XI) and (XIII); the Z or partial Z conformation appears in high salt solution for the oligomers, (II), (III), (VII), (VIII) and (IX); an A or partial A conformation appears in high salt solution for (XII). In the crystalline state, (IV), (VIII), (X), and (XI) stay in the B-form and all of the other oligomers adopt the complete Z-form except for (XII) which crystallizes in the A form. In both the crystal and in aqueous solutions, the identification of the conformation genus was made by means of Raman spectroscopy. In the crystal of (I), grown at pH7.0, guanosine is found to be in C3'-endo/syn conformation and cytidine in C2'-endo/anti, which may be taken as the ideal building block of the typical Z conformation. At pH4, (I) crystallizes in a conformation similar to the B genus. A study of the thermally induced B to Z transition has been carried out for (II) and (III). Based on the analysis of Raman spectra of the alternating pyrimidine-purine oligomers which might be expected to go into the Z form, the tendency for these oligonucleotides to adopt the Z form can be ranked as: d(CGCGCGCG) greater than (II) greater than (III) greater than (V) approximately (VI) greater than (IV) for octamers and (VII) greater than (VIII) greater than (IX) for the decamers. Similarly, those oligomers which might have a tendency to go into the A form could be ranked as (XII) greater than (XIII) approximately (X) greater than (XI). These data should provide help in formulating rules for predicting the sequence dependence of the B to A and B to Z transitions. Some possible rules are explored, but precautions should be taken.     

Sequence dependence of the B to Z transition in crystals and aqueous NaCl solutions for deoxyoligonucleotides containing all four canonical DNA bases

A laser Raman study has been made on the conformation of a series of self-complementary octameric deoxynucleotides that contain all four canonical deoxynucleotide bases [guanine (G), cytosine (C), adenine (A), and thymine (T)] in order to determine which sequences will crystallize in the Z form and which sequences will go into the Z form in aqueous solution at high salt concentrations (4-6 M NaCl). All four octadeoxynucleotides, d(TGCGCGCA) (I), d(CACGCGTG) (II), d(CGTGCACG) (III), and d(CGCATGCG) (IV), have been crystallized from low-salt solutions. The Raman spectra of microcrystals show that I, II, and IV crystallize in a rigorous Z form while III crystallizes in the B form. Sequences I and II go into a Z form in 4-6 M NaCl solution at 0 degrees C while sequences III and IV remain in the B form in 6 M salt. There are substantial differences in the Raman spectra of oligonucleotides in the Z form found in the crystal and in high-salt solutions. The Raman spectra of the Z forms in 6 M NaCl solution at 0 degrees C are not linear combinations of the Raman spectra of the complete Z form in the crystal and the complete B form in low-salt solutions. The terminal residues of these oligomers do not appear to be in a strict Z form. A detailed analysis of the ring puckers and syn/anti conformation for all of the residues both in solution and in the crystal has been made.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Prediction of atomic structure from sequence for double helical DNA oligomers

DNA can adopt different conformations depending on the base sequence, solvent, electrolyte composition and concentration, pH, temperature, and interaction with proteins. Here we present a model for calculating the three-dimensional atomic structure of double-stranded DNA oligomers. A theoretical energy function is used for calculating the interactions within the base steps and an empirical backbone function is used to restrict the conformational space accessible to the bases and to account for the conformational coupling of neighboring steps in a sequence. Conformational searching on large structures or a large number of structures is possible, because each base step can be described by just two primary degrees of freedom (slide and shift). A genetic algorithm is used to search for low-energy structures in slide-shift space, and this allows very rapid optimization of DNA oligomers. The other base step parameters have been previously optimized for all possible slide-shift sequence combinations, and a heuristic algorithm is used to add the atomic details of the backbone conformation in the final step of the calculation. The structures obtained by this method are very similar to the corresponding X-ray crystal structures observed experimentally. The average RMSD is 2.24 Angstroms for a set of 20 oligomer structures. For 15 of these sequences, the X-ray crystal structure is the global energy minimum. The other 5 are bistable sequences that have B-form global energy minima but crystallize as A-DNA.     

The high salt form of poly(dG-dC).poly(dG-dC) is left-handed Z-DNA: Raman spectra of crystals and solutions.

The laser-Raman spectra of crystalline d(CpGpCpGpCpG) and of aqueous poly(dG-dC).poly(dG-dC) in high salt (4M NaCl) and low salt (0.1M NaCl) solutions have been measured and compared. The spectra of the crystal and the high-salt solution show a striking congruence, which indicates clearly that the high-salt form of the aqueous polymer has the left-handed Z-DNA structure of the crystalline oligomer. These two spectra differ substantially from that of the low-salt form of the polymer, which has been found previously to have spectral characteristics of the B-form of DNA. The high salt spectrum shows a unique line due to guanine residues at 625 cm-1 which should be useful for qualitative and possibly quantitative assessment of the amount of Z-structure present in a sample of DNA.     

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

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

Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four‐helix bundle structure containing a covalently bound five‐coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer–monomer transition according to the absence and presence of CO. Herein, domain‐swapped dimeric AVCP was constructed and utilized to form a tetramer and high‐order oligomers. The X‐ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain‐swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain‐swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit–subunit interactions at the interfaces of the domain‐swapped dimer and monomer subunits in the tetramer were also similar to the subunit–subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain‐swapped dimer and two monomers upon CO binding. Without monomers, the domain‐swapped dimers formed tetramers, hexamers, and higher‐order oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers in the presence of CO, demonstrating that the domain‐swapped dimer maintains the CO‐induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer–monomer transition protein.     

Studies on the base pair binding specificity of CC-1065 to oligomer duplexes

In this paper, we report on the base pair binding specificity of CC-1065 to oligomer duplexes of several lengths and base composition as determined by circular dichroism (CD) methods. The oligomers are synthesized using the phosphoramidite triester coupling approach and purified by either polyacrylamide gel electrophoresis or anion-exchange HPLC. CC-1065 binds by two different mechanisms to form a reversibly bound species and an irreversibly bound species, both of which show intense induced CD bands. The reversible to irreversible binding transition is characterized by a shift of the CD band to shorter wavelength (392----371 nm) characteristic of the reaction between the cyclopropyl group of CC-1065 and the N-3 of adenine. The induced CD acquired by the CC-1065 chromophore increases with increasing oligomer chain length and with an increase in the number of bases to the 5' end of the site of attachment whether a purine or a pyrimidine is at position 5 (or 4) from the site of attachment at the 3' end is not crucial for binding. The binding sequences 5'-GATAT and 5'-GTATA show a slower conversion to an irreversibly bound species relative to the preferred sequences 5'-AAA and 5'-TTA. A G base pair at position 3 in 5'-AAGAA hinders the formation of the irreversibly bound species relative to the 5'-GAAAA and 5'-AGAAA sequences. Very stable reversible binding is observed with the sequences 5'-GAATT and 5'-AAGAA. The sequences 5'-GCGAA and 5'-AGAG also show reversible binding but are characterized by a relatively small induced molar ellipticity which decreases with time. These binding characteristics signify weaker binding for these sequences. Overall, these binding studies agree with earlier sequence studies which found two preferred binding sequences, 5'-AAAAA and 5'-PuNTTA, with CC-1065 attached to the 3' end of the binding site. Furthermore, according to studies of the oligomer 5'-CGCGAATTCGCG-3' under different experimental conditions, the annealing conditions of this work produced oligomer duplex structures, not hairpin structures. In these studies, we found that CC-1065 binds very little or not at all to hairpin structures.     

A duplex of the oligonucleotides d(GGGGGTTTTT) and d(AAAAACCCCC) forms an A to B conformational junction in concentrated salt solutions

The coexistence of both A form and B form tracts and formation of an A-B junction in the oligomer d(GGGGGTTTTT).d(AAAAACCCCC) in saturated sodium chloride solution have been detected by Raman spectroscopy. The entire duplex adopts the familiar B-form conformation in aqueous solution at low salt concentrations (0.1M NaCl). In 6M NaCl the adoption of an A form is observed within the G,C tract while a B-form is maintained in the A.T tract. The experimental results indicate that two different helical forms can co-exist in a rather short oligonucleotide and that formation of an A-B junction can occur over a fairly small span of bases. This is in agreement with recent rules governing the relation between base sequence and secondary structure of DNA published from this laboratory. The conformational preferences of each of the individual oligomers d(AAAAACCCCC) and d(GGGGGTTTTT) have also been investigated. The oligomer d(AAAAACCCCC) is single stranded but some evidence for base stacking is observed at 2 degrees C. In contrast, a double stranded B-form structure characterized by wobble G-T base pairing is observed for d(GGGGGTTTTT) in 0.1M and 6M NaCl.     

Z helix-coil transition of d(C-Br8G-C-G-C-Br8G) studied by CD, 1H-NMR and IR spectroscopies

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

Novel DNA superstructures formed by telomere-like oligomers.

DNA oligomers containing three or more contiguous guanines form tetrastranded parallel complexes, G4-DNA, in the presence of alkali cations. However, oligomers that have a single multi-guanine motif at their 3' or 5' end, with a guanine as the terminal base, also form higher order products. Thus, the oligomer T8G3T forms a unique G4-DNA product at neutral pH in the presence of Na+, K+, or Rb+; however, its isomeric counterpart T9G3 in K+ or Rb+ generates an additional ladder of products of substantially lower gel mobility. We show that these larger complexes contain, respectively, 8, 12, or 16 distinct strands of oligomer. The octamer structure formed by T9G3 assembles in moderate salt at room temperature and melts around 60 degrees C in 100 mM KCl. Methylation protection experiments suggest a nested head-to-tail superstructure containing two tetraplexes bonded front-to-back via G quartets formed by out-of-register guanines. Naturally occurring chromosomal telomeres, which all have guanines at their 3' termini, may be able to form these superstructures.     

Sequence-dependent variation in the conformation of DNA

The specificity of action of the enzyme DNAase I on double-stranded DNA polymers of defined sequence has been investigated. The results obtained with the alternating copolymers poly[d(A-T)] · poly[d(A-T)] and poly[d(G-C)] · poly[d(G-C)] support the suggestion of Klug et al. (1979) that regions of double-stranded DNA containing alternating purine-pyrimidine sequences may exist as structural variants of the classical B-form under physiological salt conditions. Digestion of defined oligomers containing alternating dG-dC sequences indicate that these too exist in some “alternating-B” structure in solution under similar conditions. The results obtained with the oligomers also provide a number of insights into the mode of action of DNAase I.In the case of the B-DNA dodecamer d(C-G-C-G-A-A-T-T-C-G-C-G), for which the crystal structure has been solved (Dickerson &; Drew, 1981), there is a very good correlation between the sites of rapid DNAase I cutting and positions of high local helical twist.     

A Duplex of the Oligonucleotides d(GGGGGTTTTT) and d(AAAAACCCCC) Forms an A to B Conformational Junction in Concentrated Salt Solutions

Abstract

The coexistence of both A form and B form tracts and formation of an A-B junction in the oligomer d(GGGGGTTTTT)· d(AAAAACCCCC) in saturated sodium chloride solution have been detected by Raman spectroscopy. The entire duplex adopts the familiar B-form conformation in aqueous solution at low salt concentrations (0.1M NaCl). In 6M NaCl the adoption of an A form is observed within the G,C tract while a B-form is maintained in the A,T tract. The experimental results indicate that two different helical forms can co-exist in a rather short oligonucleotide and that formation of an A-B junction can occur over a fairly small span of bases. This is in agreement with recent rules governing the relation between base sequence and secondary structure of DNA published from this laboratory.

The conformational preferences of each of the individual oligomers d(AAAAACCCCC) and d(GGGGGTTTTT) have also been investigated. The oligomer d(AAAAACCCCC) is single stranded but some evidence for base stacking is observed at 2°C. In contrast, a double stranded B-form structure characterized by wobble G-T base pairing is observed for d(GGGGGTTTTT) in 0.1M and 6M NaCl.     

DNA curvature and phosphate neutralization: an important aspect of specific protein binding

A theoretical study is presented of the influence of salt bridges between protein cationic side chains and DNA phosphates on DNA conformation and flexibility. Two DNA sequences are studied containing respectively the HNF3 and CAP binding sites. The effect of salt bridges is modelled by the neutralisation of net phosphate charges for the groups involved in such interactions in the complex. Energy optimised conformations are obtained by molecular mechanics calculations using the JUMNA program. Base sequence dependence is studied by moving the phosphate neutralisation pattern along the sequence, while normal mode analysis is used to evaluate DNA flexibility. The results show that phosphate neutralisation has a strong influence on DNA conformation. For the HNF3 binding sequence, the free oligomer is bent in direction very different from that observed in the protein complex. Phosphate neutralisation changes this direction by 45 degrees to within only 4 degrees of the direction in the complex, without changing the bending angle. For the CAP binding sequence, the free oligomer is already intrinsically curved in the direction favoured by the protein, but phosphate neutralisation increases the bending angle. For both oligomers studied these effects are strongly sequence dependent. It is also shown that oligomer flexibility cannot be explained by a simple superposition of the properties of successive dinucleotide steps. Important long range coupling effects are observed. However, for both sequence studied, phosphate neutralisation however leads to a reduction in oligomer flexibility.     

Prediction of protein oligomer structures using GALAXY in CASP13

Many proteins need to form oligomers to be functional, so oligomer structures provide important clues to biological roles of proteins. Prediction of oligomer structures therefore can be a useful tool in the absence of experimentally resolved structures. In this article, we describe the server and human methods that we used to predict oligomer structures in the CASP13 experiment. Performances of the methods on the 42 CASP13 oligomer targets consisting of 30 homo-oligomers and 12 hetero-oligomers are discussed. Our server method, Seok-assembly, generated models with interface contact similarity measure greater than 0.2 as model 1 for 11 homo-oligomer targets when proper templates existed in the database. Model refinement methods such as loop modeling and molecular dynamics (MD)-based overall refinement failed to improve model qualities when target proteins have domains not covered by templates or when chains have very small interfaces. In human predictions, additional experimental data such as low-resolution electron microscopy (EM) map were utilized. EM data could assist oligomer structure prediction by providing a global shape of the complex structure.     

Mapping of accessible sites for oligonucleotide hybridization on hepatitis delta virus ribozymes

Semi-random libraries of DNA 6mers and RNase H digestion were applied to search for sites accessible to hybridization on the genomic and antigenomic HDV ribozymes and their 3′ truncated derivatives. An approach was proposed to correlate the cleavage sites and most likely sequences of oligomers, members of the oligonucleotide libraries, which were engaged in the formation of RNA–DNA hybrids. The predicted positions of oligomers hybridizing to the genomic ribozyme were compared with the fold of polynucleotide chain in the ribozyme crystal structure. The data exemplified the crucial role of target RNA structural features in the binding of antisense oligonucleotides. It turned out that cleavages were induced if the bound oligomer could adapt an ordered helical conformation even when it required partial penetration of an adjacent double-stranded region. The major features of RNA structure disfavoring hybridization and/or RNase H hydrolysis were sharp turns of the polynucleotide chain and breaks in stacking interactions of bases. Based on the predicted positions of oligomers hybridizing to the antigenomic ribozyme we chose and synthesized four antisense DNA 6mers which were shown to direct hydrolysis in the desired, earlier predicted regions of the molecule.     

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.     

Circular dichroism measurements show that C.C+ base pairs can coexist with A.T base pairs between antiparallel strands of an oligodeoxynucleotide double-helix.

We have studied the coil-to-helix transition of the DNA oligomer d(C4A4T4C4), using circular dichroism measurements to monitor the formation of A.T base pairs within the central self-complementary A4T4 region and the formation of protonated C.C+ base pairs at the ends of the oligomer. We found that both A.T and C.C+ base pairs formed in a coordinated fashion as the temperature and pH were lowered. The CD data of the helix form of the oligomer were consistent with the presence of paired oligomers, but not with hairpin loops. The pKa for formation of C.C+ base pairs between the C4 ends of the oligomer was higher than the pKa for formation of C.C+ base pairs in d(C8), indicating that the formation of C.C+ base pairs in the oligomer was influenced by the presence of a paired A4T4 region. We conclude that A.T and C.C+ base pairs coexist in the self-complex of the oligomer and, therefore, that C.C+ base pairs can form between antiparallel DNA strands.