Triple helical polynucleotidic structures: an FTIR study of the C+ .G. Ctriplet.

Triple helixes containing one homopurine poly dG or poly rG strand and two homopyrimidine poly dC or poly rC strands have been prepared and studied by FTIR spectroscopy in H2O and D2O solutions. The spectra are discussed by comparison with those of the corresponding third strands (auto associated or not) and of double stranded poly dG.poly dC and poly rG.poly rC in the same concentration range and salt conditions. The triplex formation is characterized by the study of the base-base interactions reflected by changes in the spectral domain involving the in-plane double bond vibrations of the bases. Modifications of the initial duplex conformation (A family form for poly rG.poly rC, B family form for poly dG.poly dC) when the triplex is formed have been investigated. Two spectral domains (950-800 and 1450-1350 cm-1) containing absorption bands markers of the N and S type sugar geometries have been extensively studied. The spectra of the triplexes prepared starting with a double helix containing only riboses (poly rC+.poly rG.poly rC and poly dC+.poly rG.poly rC) as well as that of poly rC+.poly dG.poly dC present exclusively markers of the North type geometry of the sugars. On the contrary in the case of the poly dC+.poly dG.poly dC triplex both N and S type sugars are shown to coexist. The FTIR spectra allow us to propose that in this case the sugars of the purine (poly dG) strand adopt the S type geometry.     

Circular dichroism spectroscopy of conformers of (guanine + adenine) repeat strands of DNA

(Guanine+adenine) strands of DNA are known to associate into guanine tetraplexes, homodimerize into parallel or antiparallel duplexes, and fold into a cooperatively melting single strand resembling the protein alpha helix. Using CD spectroscopy and other methods, we studied how this conformational polymorphism depended on the primary structure of DNA. The study showed that d(GGGA)(5) and d(GGA)(7) associated into homoduplexes at low salt or in the presence of LiCl but were prone to guanine tetraplex formation, especially in the presence of KCl. In addition, they yielded essentially the same CD spectrum in the presence of ethanol as observed with the ordered single strand of d(GA)(10). Strands of d(GA)(10), d(GGAA)(5), d(GAA)(7), and d(GAAA)(5) associated into homoduplexes in both LiCl and KCl solutions, but not into guanine tetraplexes. d(GAAA)(5) and d(GAA)(7) further failed to form the single-stranded conformer in aqueous ethanol. Adenine protonation, however, stabilized the single-stranded conformer even in these adenine-rich fragments. The ordered single strands, homoduplexes as well as the guanine tetraplexes, all provided strikingly similar CD spectra, indicating that all of the conformers shared similar base stacking geometries. The increasing adenine content only decreased the conformer thermostability.     

Local effects of partial adenine-N1-oxidation on poly A-poly U and poly A-2 poly U helix conformations

We prepared helices with noncomplementary bases by N1-oxidation of poly A, followed by reaction with poly U. Mixing curves indicate that doubly and triply helical structures form, with only the unmodified adenines involved in base pair formation. Circular dichroism spectra were examined particularly at the absorbance maximum of the adenine N1-oxide (A*). In the single strand poly (A,A*), there is a relatively strong pair of positive and negative CD bands from the A*. These are greatly reduced in the double helix, and abolished in the triple helix. We conclude that A* stacks in a conventional manner with A in the single strand, but is rotated out of the double and triple helix. In the double helix the A* probably maintains a preferred orientation with respect to the helix, but rotates randomly in the triple helix.     

Structural transitions of polyadenylic acid due to protonation: the influence of the length of single strands on the polarographic behaviour of the double-helical form

Transition of single-stranded poly(A) into its double-helical protonated form was followed by means of derivative pulse polarography, spectrophotometry, and other methods. It was found that properties of protonated poly(A) depended on the length of single strands from which the protonated double helix was formed. In contrary to longer poly(A) transition of short single-stranded molecules (s₂₀,w lower than about 3) caused practically no decrease in the pulse-polarographic current. It was concluded that the formation of the protonated double helix of poly(A) did not result in the inaccesibility of the reduction sites (located in the vicinity of the surface of the molecule) for the electrode process, as it was in DNA-like double-helical polynucleotides. The current changes observed in the course of transition of longer poly(A) were explained as due to slower transport of long double-stranded molecules to the electrode.     

Enthalpy and heat capacity changes for formation of an oligomeric DNA duplex: interpretation in terms of coupled processes of formation and association of single-stranded helices.

The thermodynamics of self-assembly of a 14 base pair DNA double helix from complementary strands have been investigated by titration (ITC) and differential scanning (DSC) calorimetry, in conjunction with van't Hoff analysis of UV thermal scans of individual strands. These studies demonstrate that thermodynamic characterization of the temperature-dependent contributions of coupled conformational equilibria in the individual "denatured" strands and in the duplex is essential to understand the origins of duplex stability and to derive stability prediction schemes of general applicability. ITC studies of strand association at 293 K and 120 mM Na+ yield an enthalpy change of -73 +/- 2 kcal (mol of duplex)-1. ITC studies between 282 and 312 K at 20, 50, and 120 mM Na+ show that the enthalpy of duplex formation is only weakly salt concentration-dependent but is very strongly temperature-dependent, decreasing approximately linearly with increasing temperature with a heat capacity change (282-312 K) of -1.3 +/- 0.1 kcal K-1 (mol of duplex)-1. From DSC denaturation studies in 120 mM Na+, we obtain an enthalpy of duplex formation of -120 +/- 5 kcal (mol of duplex)-1 and an estimate of the corresponding heat capacity change of -0.8 +/- 0.4 kcal K-1 (mol of duplex)-1 at the Tm of 339 K. van't Hoff analysis of UV thermal scans on the individual strands indicates that single helix formation is noncooperative with a temperature-independent enthalpy change of -5.5 +/- 0.5 kcal at 120 mM Na+. From these observed enthalpy and heat capacity changes, we obtain the corresponding thermodynamic quantities for two fundamental processes: (i) formation of single helices from disordered strands, involving only intrastrand (vertical) interactions between neighboring bases; and (ii) formation of double helices by association (docking) of single helical strands, involving interstrand (horizontal and vertical) interactions. At 293 K and 120 mM Na+, we calculate that the enthalpy change for association of single helical strands is approximately -64 kcal (mol of duplex)-1 as compared to -210 kcal (mol of duplex)-1 calculated for duplex formation from completely unstructured single strands and to the experimental ITC value of -73 kcal (mol of duplex)-1. The intrinsic heat capacity change for association of single helical strands to form the duplex is found to be small and positive [ approximately 0.1 kcal K-1 (mol of duplex)-1], in agreement with the result of a surface area analysis, which also predicts an undetectably small heat capacity change for single helix formation.     

Sequence-targeted cleavage of nucleic acids by oligo-alpha-thymidylate-phenanthroline conjugates: parallel and antiparallel double helices are formed with DNA and RNA, respectively

Oligodeoxynucleotides can be synthesized by using the alpha anomers of nucleoside units. Oligo-alpha-deoxynucleotides are resistant to nucleases and could be used to regulate gene expression in vivo. Theoretical calculations were carried out to determine the conformational energy of an oligomeric alpha-beta duplex (dA)5.(dT)5 where the adenosine strand contains natural beta-deoxyribonucleotides and the thymidine strand contains synthetic alpha-deoxyribonucleotides. These calculations predict that in the more stable B-like conformation the two strands of the double helix should run parallel to each other whereas in the more stable A-like conformation the two strands should adopt an antiparallel orientation. In order to test these predictions 1,10-phenanthroline was covalently attached to the 5'-end of an alpha-octathymidylate. In the presence of copper ions and a reducing agent (beta-mercaptopropionic acid), the (phenanthroline)2-copper complex generates OH. radicals that cleave phosphodiester bonds in the complementary sequence to which the alpha-octathymidylate is bound. By use of a 27mer oligo-beta-deoxynucleotide containing an octadeoxyadenylate sequence as a target for the phenanthroline-substituted alpha-(dT)8, cleavage was observed on the 5'-side of the (dA)8 sequence, demonstrating that the alpha-beta DNA-DNA hybrid formed a double helix with parallel orientation of the two strands. The same result was obtained when alpha-(dT)8 was bound to beta-(dA)n with n = 8 or 10. When a beta-oligoriboadenylate was used as a target, cleavage occurred exclusively on the 3'-side of the (rA)8 or (rA)10 sequence, indicating that the alpha-beta DNA-RNA hybrid formed a double helix with an antiparallel orientation of the two strands. When a phenanthroline-substituted beta-octathymidylate was used instead of the alpha-octathymidylate, an antiparallel double helix was formed independently of whether the target beta sequence was a DNA or an RNA.     

One left-handed strand in DNA-oligonucleotide complexes?

A single strand of oligonucleotide can bind to double helical DNA under certain conditions. This must involve some unwinding of the original double helix in a process leading to the formation of a three-stranded region. The free energy for such an entropically unlikely reaction may come from a change in the degree of supercoiling of the original DNA. The conformation of the triple strand is investigated here using computer graphics and molecular mechanics calculations. It is suggested that on binding the oligonucleotide (strand 3) to two paired strands (1 and 2) in a supercoiled DNA molecule, strand 2 might adopt a left-handed conformation whilst strand 1 and strand 3 pair in the normal Watson-Crick B-configuration.     

Mechanisms of DNA binding determined in optical tweezers experiments

The last decade has seen rapid development in single molecule manipulation of RNA and DNA. Measuring the response force for a particular manipulation has allowed the free energies of various nucleic acid structures and configurations to be determined. Optical tweezers represent a class of single molecule experiments that allows the energies and structural dynamics of DNA to be probed up to and beyond the transition from the double helix to its melted single strands. These experiments are capable of high force resolution over a wide dynamic range. Additionally, these investigations may be compared with results obtained when the nucleic acids are in the presence of proteins or other binding ligands. These ligands may bind into the major or minor groove of the double helix, intercalate between bases or associate with an already melted single strand of DNA. By varying solution conditions and the pulling dynamics, energetic and dynamic information may be deduced about the mechanisms of binding to nucleic acids, providing insight into the function of proteins and the utility of drug treatments.     

A new model for the K+-induced macromolecular structure of guanosine 5'-monophosphate in solution.

The (31)P NMR spectra of (TMA)(2)(5'-GMP), where TMA is [(CH(3))(4)N](+) and 5'-GMP is guanosine 5'-monophosphate, and K(2)(5'-GMP), containing various amounts of KCl or TMACl, have been obtained at 2 degrees C. Variable-temperature spectra have also been obtained for K(2)(5'-GMP). The TMA(+) ion serves to neutralize the charge on the dianionic 5'-GMP and permits the added K(+) to bond preferentially in structure-forming sites. (1)H NMR spectra (one- and two-dimensional) have been obtained for K(2)(5'-GMP) and used to assign the proton resonances in the self-associated structures and determine that all residues have the anti glycosidic conformation. The (31)P and (1)H NMR spectra are very complex and indicate the presence of a large number of molecular environments and a structural variation dependent upon the mole ratio of 5'-GMP to K(+). A new model for the solution structure is proposed in which the 5'-GMP forms a pseudo-four-stranded helix with guanine-guanine hydrogen bonding forming a continuous helical strand, rather than the usual planar G-tetrad structure. The guanine-guanine hydrogen bonding sites are the same as that found in a G-tetrad. The K(+) ions would be located in the center of the helix and bonding to the carbonyl oxygens. They are interacting with the phosphates as well. Integration data from the largest sized species give an estimate of 14.3 +/- 1.1 residues in a helical structure.     

A proposal for a specific double-helical structure in which the polynucleotide strands intercalate instead of forming base-pairs

Double helices, since the discovery of the DNA structure by Watson and Crick, represent the single most important secondary structural form of nucleic acids. The secondary structures of a variety of polynucleotide helices have now been well characterised with hydrogen-bonded base-pairs as building blocks. We wish to propose here the possibility, in a specific case, of a double stranded helical structure without any base-pair, but having a repeat unit of two nucleotides with their bases stacked through intercalation. The proposal comes from the initial models we have built for poly(dC) using the stacking patterns found in the crystal structures of 5'-dCMPNa2 which crystallises in two forms depending on the degree of hydration. These structures have pairs of nucleotides with the cytosine rings partially overlapping and separated by 3.3A. Using these as repeat units one could generate a model for poly(dC) with parallel strands, having a turn angle of 30 degrees and a base separation of 6.6A along each strand. Both right and left handed models with these parameters can be built in a smooth fashion without any obviously unreasonable stereochemical contacts. The helix diameter is about 13.5A, much smaller than that of normal helices with base-pair repeats. The changes in the sugar-phosphate backbone conformation in the present models compared to normal duplexes only reflect the torsional flexibility available for extension of polynucleotide chains as manifested by the crystal structures of drug-inserted oligonucleotide complexes. Intercalation proposed here could have some structural relevance elsewhere, for instance to the base-mismatched regions on the double helix and the packing of noncomplementary single strands as found in the filamentous bacteriophage Pf1.     

Dimerization of the guanine-adenine repeat strands of DNA.

Jovin and co-workers have demonstrated that DNA strands containing guanine-adenine repeats generate a parallel-stranded homoduplex. Here we propose that the homoduplex is a dimer of the ordered single strand discovered by Fresco and co-workers at acid pH. The Fresco single strand is shown here to be stabilized in aqueous ethanol where adenine is not protonated. Furthermore, we demonstrate that the strands dimerize at higher salt concentrations without significantly changing their conformation, so that the dimerization is non-cooperative. Hence, the Jovin homoduplex can form through a non-cooperative dimerization of two cooperatively melting single strands. The available data indicate that the guanines stabilize the Fresco single strand whereas the adenines cause dimerization owing to their known intercalation or clustering tendency. The guanine-adenine repeat dimer seems to be a DNA analog of the leucine zipper causing dimerization of proteins.     

Molecular dynamics simulations of excimer forming (+)-anti-BPDE-DNA adducts in aqueous solution

The chemical carcinogen (+)-anti BPDE preferentially binds covalently to the guanine base in the minor groove of DNA. Fluorescence spectroscopic studies have shown that the BPDE molecules bound to DNA can interact in their photo-excited state giving strong excimer fluorescence when bound to poly(dGdC) · poly(dGdC). It was suggested that the formation of such excited state complexes is most probable when the two (+)-anti-BPDE bind to guanines of adjacent base pairs on the two different strands of the DNA. In the present work a model for such an excimer forming DNA-BPDE double adduct system has been constructed and shown to be stable over a 300 ps molecular dynamics simulation in a water box. The model is a d(CG)₃ · d(CG)₃ molecule with two BPDE molecules bound to the guanines at the 4th position on each strand, located in the minor groove and each oriented towards the 5 end of the modified strand, respectively. The results of 300 ps MD simulation show that the two BPDE chromophores exhibited on the average a relative geometry favourable for excimer formation. The local structure at the adduct position was considerably distorted and the helix axis was bent. The modified bases were found to be paired through a stable single non-Watson Crick type of hydrogen bond. Correspondence to: A. Gräslund     

Nuclear magnetic resonance structural studies of intramolecular purine.purine.pyrimidine DNA triplexes in solution. Base triple pairing alignments and strand direction

Recently, P.A. Beal and P.B. Dervan, expanding on earlier observations by others, have established the formation of purine.purine.pyrimidine triple helices stabilized by G.GC, A.AT and T.AT base triples where the purine-rich third strand was positioned in the major groove of the Watson-Crick duplex and anti-parallel to its purine strand. The present nuclear magnetic resonance (n.m.r.) study characterizes the base triple pairing alignments and strand direction in a 31-mer deoxyoligonucleotide that intramolecularly folds to generate a 7-mer (R/Y-)n.(R+)n(Y-)n triplex with the strands linked by two T5 loops and stabilized by potential T.AT and G.GC base triples. (R and Y stand for purine and pyrimidine, respectively, while the signs establish the strand direction.) This intramolecular triplex gives well-resolved exchangeable and non-exchangeable proton spectra with Li+ as counterion in aqueous solution. These studies establish that the T1 to C7 pyrimidine and the G8 to A14 purine strands are anti-parallel to each other and align through Watson-Crick A.T and G.C pair formation. The T15 to G21 purine-rich third strand is positioned in the major groove of this duplex and pairs through Hoogsteen alignment with the purine strand to generate T.AT and G.GC triples. Several lines of evidence establish that the thymidine and guanosine bases in the T15 to G21 purine-rich third strand adopt anti glycosidic torsion angles under conditions where this strand is aligned anti-parallel to the G8 to A14 purine strand. We have also recorded imino proton n.m.r. spectra for an (R-)n.(R+)n(Y-)n triplex stabilized by G.GC and A.AT triples through intramolecular folding of a related 31-mer deoxyoligonucleotide with Li+ as counterion. The intramolecular purine.purine.pyrimidine triplexes containing unprotonated G.GC, A.AT and T.AT triples are stable at basic pH in contrast to pyrimidine.purine.pyrimidine triplexes containing protonated C+.GC and T.AT triples, which are only stable at acidic pH.     

High-resolution NMR study of a synthetic DNA-RNA hybrid dodecamer containing the consensus pribnow promoter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG)

The nonsymmetrical double-helical hybrid dodecamer d(CGTTATAATGCG).r(CGCAUUAUAACG) was synthesized with solid-phase phosphoramidite methods and studied by high-resolution 2D NMR. The imino protons were assigned by one-dimensional nuclear Overhauser methods. All the base protons and H1', H2', H2", H3', and H4' sugar protons of the DNA strand and the base protons, H1', H2', and most of the H3'-H4' protons of the RNA strand were assigned by 2D NMR techniques. The well-resolved spectra allowed a qualitative analysis of relative proton-proton distances in both strands of the dodecamer. The chemical shifts of the hybrid duplex were compared to those of the pure DNA double helix with the same sequence (Wemmer et al., 1984). The intrastrand and cross-strand NOEs from adenine H2 to H1' resonances of neighboring base pairs exhibited characteristic patterns that were very useful for checking the spectral assignments, and their highly nonsymmetric nature reveals that the conformations of the two strands are quite different. Detailed analysis of the NOESY and COSY spectra, as well as the chemical shift data, indicate that the RNA strand assumes a normal A-type conformation (C3'-endo) whereas the DNA strand is in the general S domain but not exactly in the normal C2'-endo conformation. The overall structure of this RNA-DNA duplex is different from that reported for hybrid duplexes in solution by other groups (Reid et al., 1983a; Gupta et al., 1985) and is closer to the C3'-endo-C2'-endo hybrid found in poly(dA).poly(dT) and poly(rU).poly(dA) in the fiber state (Arnott et al., 1983, 1986).     

NMR characterization of clustered bistrand abasic site lesions: effect of orientation on their solution structure

A unique characteristic of ionizing radiation and radiomimetic anticancer drugs is the induction of clustered damage: two or more DNA lesions (oxidized bases, abasic sites, or strand breaks) occurring in the same or different strands of the DNA molecule within a single turn of the helix. In spite of arising at a lower frequency than single lesions, clustered DNA damage represents an exotic challenge to the repair systems present in the cells and, in some cases, these lesions may escape detection and/or processing. To understand the structural properties of clustered DNA lesions we have prepared two oligodeoxynucleotide duplexes containing adjacent tetrahydrofuran residues (abasic site analogues), positioned one in each strand of the duplex in a 5' or 3' orientation, and determined their solution structure by NMR spectroscopy and molecular dynamics simulations. The NMR data indicate that both duplex structures are right-handed helices of high similarity outside the clustered damage site. The thermal stability of the duplexes is severely reduced by the presence of the abasic residues, especially in a 5' orientation where the melting temperature is 5 degrees C lower. The structures show remarkable differences at the lesion site where the extrahelical location of the tetrahydrofuran residues in the (AP)(2)-5'-staggered duplex contrasts with their smooth alignment along the sugar-phosphate backbone in the (AP)(2)-3'-staggered duplex.     

A combinatorial system to examine the enzymatic repair of multiply damaged DNA substrates

DNA damage drives genetic mutations that underlie the development of cancer in humans. Multiple pathways have been described in mammalian cells which can repair this damage. However, most work to date has focused upon single lesions in DNA. We present here a combinatorial system which allows assembly of duplexes containing single or multiple types of damage by ligating together six oligonucleotides containing damaged or modified bases. The combinatorial system has dual fluorescent labels allowing examination of both strands simultaneously, in order to study interactions or competition between different DNA repair pathways. Using this system, we demonstrate how repair of oxidative damage in one DNA strand can convert a mispaired T:G deamination intermediate into a T:A mutation. We also demonstrate that slow repair of a T:G mispair, relative to a U:G mispair, by the human methyl-binding domain 4 DNA glycosylase provides a competitive advantage to competing repair pathways, and could explain why CpG dinucleotides are hotspots for C to T mutations in human tumors. Data is also presented that suggests repair of closely spaced lesions in opposing strands can be repaired by a combination of short and long-patch base excision repair and simultaneous repair of multiply damage sites can potentially lead to lethal double strand breaks.     

Hydration of the dTn.dAn x dTn parallel triple helix: a Fourier transform infrared and gravimetric study correlated with molecular dynamics simulations.

We present a comparative analysis of the water organization around the dTn.dAn x dTn triple helix and the Watson-Crick double helix dTn.dAn respectively by means of gravimetric measurements, infrared spectroscopy and molecular dynamics simulations. The hydration per nucleotide determined by gravimetric and spectroscopic methods correlated with the molecular dynamics simulations shows that at high relative humidity (98% RH) the triple helix is less solvated than the duplex (17 +/- 2 water molecules per nucleotide instead of 21 +/-1). The experimental desorption curves are different for both structures and indicate that below 81% RH the triplex becomes more hydrated than the duplex. At this RH the FTIR spectra show the emergence of N-type sugars in the adenosine strand of the triplex. When the third strand is bound in the major groove of the Watson-Crick duplex molecular dynamics simulations show the formation of a spine of water molecules between the two thymidine strands.     

Evidence from cauliflower mosaic virus virion DNA for additional discontinuities in the plus strand.

Two-dimensional electrophoresis of cauliflower mosaic virus (CaMV) virion DNA and analysis of Southern blots using (+) strand-specific probes to the 5' termini of the beta (5.4 Kb) and alpha (2.6 Kb) strands, revealed the presence of molecules in addition to those predicted from the known structure of CaMV DNA. The presence of 8 Kb molecules of (+) sense after denaturation suggested that a small proportion of circular molecules have only a single discontinuity in the (+) strand. Other molecules, probably 5' coterminal with the beta strand but smaller than 5.4 Kb, indicated that a minority of the circular full length CaMV DNA contain additional gaps in the (+) strand. Consequently, molecules equivalent to the remainder of the beta strand could be identified using a single strand probe for a region towards the 3'-end of the beta strand. Computer analysis of the nucleotide sequence of CaMV DNA in the region of the proposed additional discontinuities revealed regions displaying some homology with the major (+) strand priming sites at the 5' ends of the beta and alpha strands. It is our contention that the additional (+) strand molecules of beta specificity are a consequence of minor (+) strand priming sites.