Vibrational normal modes and dynamical stability of DNA triplex poly(dA). 2poly(dT): S-type structure is more stable and in better agreement with observations in solution

A normal-mode and statistical mechanical calculation was carried out to determine the vibrational normal modes, contribution of internal fluctuations to the free energy, and hydrogen bond disruption of DNA triplex poly(dA).2poly(dT). The calculation was performed on both the x-ray fiber diffraction model with a N-type sugar conformation, and a newly proposed model with a S-type sugar conformation. Our calculated normal modes for the S-type structure are in better agreement with observed IR spectra for samples in D2O solution. We also find that the contribution of internal fluctuations to free energy, premelting hydrogen bond disruption probability, and hydrogen bond melting temperatures for the Hoogsteen and Watson-Crick hydrogen bonds all show that the S-type structure is dynamically more stable than the N-type structure in a nominal solution environment. Therefore our calculation supports experimental findings that the triplex d(T)n.d(A)nd(T)n most likely adopts a S-type sugar conformation in solution or at high humidity. Our calculations, however, do not preclude the possibility of an N-type conformation at lower humidities.     

Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA

Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs.     

Poly(L-lysine)-graft-dextran copolymer is a novel stabilizer of triplex DNA (I): stabilization of poly(dA).2poly(dT) triplex

Comb-type polylysine copolymer having grafted hydrophilic side chains was newly designed as a novel stabilizer of triplex DNAs. The comb-type copolymer elevated melting temperature of poly(dA).2poly(dT) triplex by 50 degrees C without affecting reversibility, melting and reassociation, of the triplex in buffer with physiological salt concentrations. The stabilizing effect of the copolymer was greater than spermine. Our results indicate that the molecular designing of polycation with comb-type structure is a successful strategy for creating an effective triplex stabilizer.     

Stability of triple-helical poly(dT)-poly(dA)-poly(dT) DNA with counterions

Structural conformation of triple-helical poly(dT)-poly(dA)-poly(dT) has been a very controversial issue recently. Earlier investigations, based on fiber diffraction data and molecular modeling, indicated an A-form conformation with C'3-endo sugar pucker. On the other hand, Raman, solution infrared spectral, and NMR studies show a B-form structure with C'2-endo sugars. In accordance with these experimental results, a theoretical model with B-form, C'2-endo sugars was proposed in 1993. In the present work we investigate the dynamics and stability of the two conformations within the effective local field approach applied to the normal mode calculations for the system. The presence of counterions was explicitly taken into account. Stable equilibrium positions for the counterions were calculated by analyzing the normal mode dynamics and free energy of the system. The breathing modes of the triple helix are shifted to higher frequencies over those of the double helix by 4-16 cm-1. The characteristic marker band for the B conformation at 835 cm-1 is split up into two marker bands at 830 and 835 cm-1. A detailed comparison of the normal modes and the free energies indicates that the B-form structure, with C'2-endo sugar pucker, is more stable than the A-form structure. The normal modes and the corresponding dipole moments are found to be in close agreement with recent spectroscopic findings.     

Synergistic effects in the melting of DNA hydration shell: melting of the minor groove hydration spine in poly(dA).poly(dT) and its effect on base pair stability

We propose that water of hydration in contact with the double helix can exist in several states. One state, found in the narrow groove of poly(dA).poly(dT), should be considered as frozen to the helix, i.e., an integral part of the double helix. We find that this enhanced helix greatly effects the stability of that helix against base separation melting. Most water surrounding the helix is, however, melted or disassociated with respect to being an integral part of helix and plays a much less significant role in stabilizing the helix dynamically, although these water molecules play an important role in stabilizing the helix conformation statically. We study the temperature dependence of the melting of the hydration spine and find that narrow groove nonbonded interactions are necessary to stabilize the spine above room temperature and to show the broad transition observed experimentally. This calculation requires that synergistic effects of nonbonded interactions between DNA and its hydration shell affect the state of water-base atom hydrogen bonds. The attraction of waters into narrow groove tends to retain waters in the groove and compress or strain these hydrogen bonds.     

Regulation of Na+/H+ exchanger gene expression. Role of a novel poly(dA.dT) element in regulation of the NHE1 promoter

In this study we examine regulation of expression of the Na+/H+ exchanger promoter in L6 and NIH 3T3 cells. We have identified a highly conserved poly(dA dT)-rich region that appears to be important in regulation of expression of the NHE1 gene. Deletion or mutation of this region results in dramatic decreases in promoter activity in both L6 and NIH 3T3 cells. In addition, DNase I footprinting experiments demonstrated that this region is protected by nuclear extracts from both cell types, and gel mobility shift assays showed that a protein or proteins specifically binds to the poly(dA dT)-rich element. Using Southwestern blotting, we determined that a 33-kDa protein binds to the poly(dA dT)-containing region. Mutations that abolished protein binding to this element diminished activity of the promoter. Insertion of the poly(dA dT)-rich element into a plasmid containing the SV40 promoter demonstrated that this element can also enhance the activity of a foreign promoter. Together, the results we have presented here show that the poly(dA dT)-rich region is important in regulation of NHE1 expression in different cell types.     

Interactions of the antiviral quinoxaline derivative 9-OH-B220 [2, 3-dimethyl-6-(dimethylaminoethyl)- 9-hydroxy-6H-indolo-[2, 3-b]quinoxaline] with duplex and triplex forms of synthetic DNA and RNA

The binding of an antiviral quinoxaline derivative, 2,3-dimethyl- 6 - (dimethylaminoethyl) - 9 - hydroxy - 6H - indolo - [2,3 - b]quinoxaline (9-OH-B220), to synthetic double and triple helical DNA (poly(dA).poly(dT) and poly(dA).2poly(dT)) and RNA (poly(rA). poly(rU) and poly (rA).2poly(rU)) has been characterized using flow linear dichroism (LD), circular dichroism (CD), fluorescence spectroscopy, and thermal denaturation. When either of the DNA structures or the RNA duplex serve as host polymers a strongly negative LD is displayed, consistent with intercalation of the chromophoric ring system between the base-pairs/triplets of the nucleic acid structures. Evidence for this geometry also includes weak induced CD signals and strong increments of the fluorescence emission intensities upon binding of the drug to each of these polymer structures. In agreement with intercalative binding, 9-OH-B220 is found to effectively enhance the thermal stability of both the double and triple helical states of DNA as well as the RNA duplex. In the case of poly(dA).2poly(dT), the drug provides an unusually large stabilization of its triple helical state; upon binding of 9-OH-B220 the triplex-to-duplex equilibrium is shifted towards higher temperature by 52.5 deg. C in a 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA. When triplex RNA serves as host structure, LD indicates that the average orientation angle between the drug chromophore plane and the helix axis of the triple helical RNA is only about 60 to 65 degrees. Moreover, the thermal stabilizing capability, as well as the fluorescence increment, CD inducing power and perturbations of the absorption envelope, of 9-OH-B220 in complex with the RNA triplex are all less pronounced than those observed for the complexes with DNA and duplex RNA. These features indicate binding of 9-OH-B220 in the wide and shallow minor groove of poly(rA).2poly(rU). Based on the present results, some implications for the applications of this low-toxic, antiviral and easily administered drug in an antigene strategy, as well as its potential use as an antiretroviral agent, are discussed.     

Orthodontic treatment and temporomandibular disorders

Resonance Raman spectra excited at 257 nm are reported for the complexes of the Nickel, Cobalt and Zinc derivatives of Tetrakis(4-N-methylpyridyl)porphine with poly(dA.dT)2, poly(dA).poly(dT), poly(dG.dC)2 and poly(dG).poly(dC). These spectra are interpreted as evidence of multiple outside binding modes with poly(dA).poly(dT), and of evidence for an outside binding mode with Poly(dG.dC). Some results obtained for the zinc derivative with poly(dA).poly(dT) suggest a binding mode peculiar to this derivative.     

Coralyne binds tightly to both T.A.T- and C.G.C(+)-containing DNA triplexes

Coralyne is a DNA-binding antitumor antibiotic whose structure contains four fused aromatic rings. The interaction of coralyne with the DNA triplexes poly(dT).poly(dA).poly(dT) and poly[d(TC)].poly[d(GA)].poly[d(C+T)] was investigated by using three techniques. First, Tm values were measured by thermal denaturation analysis. Upon binding coralyne, both triplexes showed Tm values that were increased more than those of the corresponding duplexes. A related drug, berberinium, in which one of the aromatic rings is partially saturated, gave much smaller changes in Tm. Second, the fluorescence of coralyne is quenched in the presence of DNA, allowing the measurement of binding parameters by Scatchard analysis. The binding isotherms were biphasic, which was interpreted in terms of strong intercalative binding and much weaker stacking interactions. In the presence of 2 mM Mg2+, the binding constants to poly(dT).poly-(dA).poly(dT) and poly[d(TC)].poly[d(GA)].poly[(C+T)] were 3.5 x 10(6) M-1 and 1.5 x 10(6) M-1, respectively, while the affinity to the parent duplexes was at least 2 orders of magnitude lower. In the absence of 2 mM Mg2+, the binding constants to poly[d(TC)].poly[d(GA)].poly[d(C+T)] and poly-[d(TC)].poly[d(GA)] were 40 x 10(6) M-1 and 15 x 10(6) M-1, respectively. Thus coralyne shows considerable preference for the triplex structure but little sequence specificity, unlike ethidium, which will only bind to poly(dT).poly(dA).poly(dT). Further evidence for intercalation of coralyne was provided by an increase in the relative fluorescence quantum yield at 260 nm upon binding of coralyne to triplexes as well as an absence of quenching of fluorescence in the presence of Fe[(CN)6]4-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA). poly (dT) method

The hybrid plasmid PBETAG, consisting of plasmid PMB9 DNA with an insert of rabbit globin DNA (about 600 base pairs) flanked by poly(dA) poly(dT) regions (Maniatis, T., Kee, S.G., Efstratiadis, A. and Kafatos, F.C. (1976) Cell 8, 163-182), was cleaved into two fragments by endonuclease S1 under conditions of partial denaturation. Only the smaller fragment (575 base pairs) contained globin-specific sequences, showing that excision had occurred in the A-T-rich regions. This method of cleavage provides a useful procedure for assessing the length of inserts in hybrid plasmids prepared by the poly(dA)-POLY(DT) tail method, and allows the preparative recovery of the insert.     

Molecular dynamics investigations of DNA triple helical models: unique features of the Watson-Crick duplex

We have built computer models of triple helical structures with a third poly(dT) strand Hoogsteen base paired to the major groove of a poly(dA).poly(dT) Watson-Crick (WC) base-paired duplex in the canonical A-DNA as well as B-DNA. For the A-DNA form, the sugar-phosphate backbone of the third strand intertwines and clashes with the poly(dA) strand requiring a radical alteration of the duplex to access the hydrogen bonding sites in the major groove. In contrast, when the duplex was in the canonical B-DNA form, the third strand was readily accommodated in the major groove without perturbing the duplex. The triple helical model, with the duplex in the B-DNA form, was equilibrated for 400ps using molecular dynamics simulations including water molecules and counter-ions. During the entire simulations, the deoxyriboses of the adenine strand oscillate between the S-type and E-type conformations. However, 30% of the sugars of the thymine strands-II & III switch to the N-type conformation early in the simulations but return to the S-type conformation after 200ps. In the equilibrium structure, the WC duplex portion of the triplex is unique and its geometry differs from both the A- or B-DNA. the deoxyriboses of the three strands predominantly exhibit S-type conformation. Besides the sugar pucker, the major groove width and the base-tilt are analogous to B-DNA, while the X-displacement and helical twist resemble A-DNA, giving a unique structure to the triplex and the Watson & Crick and Hoogsteen duplexes.     

Probing of DNA-binding sites of Escherichia coli RecA protein utilizing 1-anilinonaphthalene-8-sulfonic acid

RecA protein of Escherichia coli plays an essential role in homologous recombination of DNA strands. To analyze the interaction of RecA with single-stranded DNA (ssDNA), we performed a fluorescence competition assay employing 1-anilinonaphthalene-8-sulfonic acid (ANS) as an extrinsic fluorescent probe. ANS bound to RecA at three sites, leading to enhancement of ANS fluorescence. Addition of synthetic polynucleotides to the RecA-ANS complex in the absence of a nucleotide quenched the ANS fluorescence, indicating displacement of ANS molecules by ssDNA. Less effective quenching by poly(dA) suggests that the nucleoprotein filament on poly(dA) may differ from those on poly(dT) and poly(dC). A titration experiment with poly(dT) and poly(dA) showed clear stoichiometric binding of 3.5 nucleotides per protein. The site size for poly(dC) was 7.0, which could be explained by the formation of a double helix of poly(dC). ATP and other nucleotides also displaced the ANS. To identify ANS-binding sites, ANS was incorporated into RecA by UV irradiation, and fluorescent peptides were isolated from the proteolytic digest. Sequence analysis suggested that ANS binds to or near the ATP-binding region. These results suggest that the fluorescence quenching and photoincorporation assay using ANS may be useful for the analysis of the interaction of a protein and its ligand.     

Effects of hydration, ion release, and excluded volume on the melting of triplex and duplex DNA

The stability of DNA duplex and triplex structures not only depends on molecular forces such as base pairing or tripling or electrostatic interactions but also is sensitive to its aqueous environment. This paper presents data on the melting of Escherichia coli and poly(dA).poly(dT) duplex DNA and on the poly(dT).poly(dA). poly(dT) triplex in a variety of media to assess the contributions from the osmotic status and salt content of the media. The effects of volume exclusion on the stability of the DNA structures are also studied. From thermal transition measurements in the presence of low-molecular weight osmotic stressors, the number of water molecules released upon melting is found to be four waters per base pair for duplex melting and one water for the conversion of triplex to single-strand and duplex. The effects of Na+ counterion binding are also determined in ethylene glycol solutions so that the variation of counterion binding with water activity is evaluated. The data show that there is a modest decrease in the extent of counterion binding for both duplex and triplex as water activity decreases. Finally, using larger polyethylene glycol cosolutes, the effects on melting of volume exclusion by the solutes are assessed, and the results correlated with simple geometric models for the excluded volume. These results point out that DNA stability is sensitive to important conditions in the environment of the duplex or triplex, and thus, conformation and reactivity can be influenced by these solution conditions.     

Different effects of nonintercalative antitumor drugs on DNA triple helix stability: SN-18071 promotes triple helix formation

The interaction of the nonintercalating bisquaternary ammonium heterocyclic drugs SN-18071 and SN-6999 with a DNA triple helix has been studied using thermal denaturation and CD spectroscopy. Our data show, that both minor groove binders can bind to the triple helix of poly(dA).2poly(dT) under comparable ionic conditions, but they influence the stability of the triplex relative to the duplex structure of poly(dA).poly(dT) in a different manner. SN-18071, a ligand devoid of forming hydrogen bonds, can promote triplex formation and thermally stabilizes it up to 500 mM Na+ concentration. SN-6999 destabilizes the triplex to duplex equibilirium whereas it stabilizes the duplex. The binding constant of SN-18071 is found to be greater than that to the duplex. The stabilizing effect of SN-18071 is explained by electrostatic interactions of three ligand molecules with the three grooves of the triple stranded structure. From the experiments it is concluded that SN-6999 binds to the triplex minor groove thereby destabilizing the triplex similar as previously reported for netropsin.     

Ligands with affinity to specific sequences of DNA base pairs. X. Synthesis and binding of netropsin analogs, containing a chelating copper ion peptide, with DNA

An analogue of netropsin has been synthesized consisting of two N-propylpyrrolcarboxamide units linked covalently to a copper-chelating tripeptide Gly-Gly-L-His by means of two and three glycine residues. Binding to DNA and synthetic polynucleotides of netropsin analogue containing three glycine residues between Gly-Gly-L-His tripeptide and the N-end of netropsin analogue (His-Nt) has been studied. It is shown that this netropsin analogue chelates a copper ion with 1:1 stoichiometry, similar to a free Gly-Gly-L-His peptide. It is found that this netropsin analogue occupies 3 to 4 base pairs upon binding to poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers, irrespective of whether it binds in Cu(2+)-ligated or unligated forms. Binding constants and binding site sizes have been calculated for netropsin analogue complexes with DNA, poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers at the [Cu2+]/[His-Nt] ratio equal to 0 and 1.0. In the three-component system including His-Nt and Cu(2+)-His-Nt, cooperative effects are recognized which can be explained by heterodimer generation on interaction of His-Nt and Cu(2+)-His-Nt at adjacent binding sites.     

Detection of the sites of alkylation in DNA and polynucleotides by laser Raman spectroscopy

A laser Raman study of the alkylation of calf thymus DNA, poly(dG)-poly(dC) and poly(dA)-(dT) has been made using two water soluble alkylating agents: an antitumor drug, the difunctional methyl nitrogen mustard (HN2), which froms interstrand cross-links, and the dimethyl nitrogen half mustard (HN1). When an excess of the alkylating agent was used, the observed Raman frequencies due to the guanine ring modes in DNA and poly(dG)-poly(dC) changed virtually quantitatively to those of 7-methylguanosine (7-Me-Guo) showing that essentially all of the guanine bases were alkylated in the N-7 position. Furthermore, this alkylated DNA formed a stable double helical complex at neutral pH in which the alkylated guanine residues are in the keto form. No changes in the Raman bands of any of the other bases were observed in alkylated DNA. The DNA double helix, completely alkylated in at the N-7 position of guanine, melts about 35 degrees C below that of the native DNA. Upon melting, the alkylated guanine changes from the keto to the zwitterionic form.     

Structure of poly d(AI).poly d(CT) in two different packing arrangements

X-ray diffraction analysis of poly d(AI).poly d(CT) in oriented and polycrystalline fibers has revealed the DNA structure to be a 10-fold, right-handed, antiparallel, Watson-Crick base paired double helix in two distinct packing arrangements corresponding to one and two helices, respectively, in the unit cell. The helix pitch is 32.1 A and 32.4 A in the two cases, almost 1.5 A shorter than in classical B-DNA. The resulting B'-DNA geometry, described in terms of a tetranucleotide repeat which is conformationally similar to B-DNA, has its minor groove closely shut and major groove correspondingly widened, thus striking a sharp morphological contrast to B-DNA. According to difference electron density maps, a spine of hydration along the minor groove connects both strands and provides structural stability; ordered sodium ions and water molecules are actively involved in bridging the phosphate groups of neighboring helices. The crystallographic R-values for these two allomorphs are 0.26 and 0.20, respectively, for data up to 3.0 A resolution.     

Termination within oligo(dT) tracts in template DNA by DNA polymerase gamma occurs with formation of a DNA triplex structure and is relieved by mitochondrial single-stranded DNA-binding protein

Xenopus laevis DNA polymerase gamma (pol gamma) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol gamma to extend a primer through a defined oligo(dT) tract. pol gamma terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T.A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol gamma. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol gamma unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol gamma to successfully replicate through dT-rich sequences.     

Change in capsulorhexis size with four foldable loop-haptic lenses over 6 months

The interaction of a series of potent leishmanicidal aromatic diamidines resembling pentamidine, was studied with Leishmania infantum DNA and polynucleotides. The diamidines viz., CGP040215A, CGP033829A and CGP039937A, interacted with leishmania DNA as well as with the polynucleotides poly(dA)-poly(dT), poly(dA-dT) and poly(dG-dC). The thermodynamic analysis to determine the association constants and the binding enthalpy pointed toward binding of the diamidines at AT regions of the DNA. The results also indicate that the diamidines bind at the outside of the DNA double helix, probably to the minor groove regions, with hydrogen bonds connecting the amide nitrogen of the diamidine to carbonyl oxygen atoms of thymidine or adenosine bases. However, CGP040215A and CGP033829A, the bisaryl diamidines, showed higher affinity than CGP039937A, the monoaryl diamidine. The spectrophotometric analysis of the interaction of these diamidines to test their effects on the melting temperature of leishmanial DNA suggests non-intercalating binding. The diamidines also showed potent inhibition of DNA polymerase activity of L. infantum extracts in vitro.     

Gene V protein dimerization and cooperativity of binding of poly(dA)

Gene V protein of bacteriophage f1 is a dimeric protein that binds cooperatively to single-stranded nucleic acids. In order to determine whether a monomer-dimer equilibrium has an appreciable effect upon the thermodynamics of gene V protein binding to nucleic acids, the dissociation constant for the protein dimer was investigated using size-exclusion chromatography. At concentrations ranging from 5 x 10(-10) to 1.2 x 10(-5) M, the Stokes radius of the protein was that expected of the dimer of the gene V protein. The Stokes radius of the protein was also independent of salt concentration from 0.2 to 1.0 M NaCl in a buffer containing 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA. The binding of the dimeric gene V protein to poly(dA) was studied using a simplified lattice model for protein-protein interactions adapted for use with a dimeric protein that binds simultaneously to two strands of nucleic acid. Interpretation of the salt dependence, C = [d log(Kint omega)]/[d log(NaCl)], of binding of such a dimeric protein to nucleic acid using the theory of Record et al. (Record, M. T., et al. (1976) J. Mol. Biol. 107, 145-158) indicates that C is a function of the numbers of cations and anions released from protein and nucleic acid upon binding of the dimer, not of the monomer. Cooperativity of gene V protein binding to poly(dA) was studied with titration experiments that are sensitive to the degree of cooperativity of binding. The cooperativity factor omega, defined as the ratio of the binding constant for a site adjacent to a previously bound dimer to that for an isolated site, was found to be relatively insensitive to salt, with a value in the range of 2000-7000 for binding to poly(dA) at 3 degrees C and at 23 degrees C. This high cooperativity factor supports the suggestion that protein-protein contacts play a major role in the formation of the superhelical gene V protein-single-stranded nucleic acid complex.