Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

Modification of duplex poly(G).poly(C) by platinum (II) compounds.

The modification of the double-stranded poly(G).poly(C) complex by cis-diamminedichloroplatinum(II) was studied by two modes: the action of cis-DDP on poly(G) before formation of the duplex with poly(C) and that on the prepared duplex. It was shown that in the latter case modification disordered the integrity of the duplex only negligibly at rb less than or equal to 0.05 and led to improved interferon-inducing and antiviral activity tested on mice infected by Influenza and Herpes viruses.     

Binding mode of 4',6-diamidino-2-phenylindole and ethidium to poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex

Optical spectroscopic properties of 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide complexed with poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex were compared in this study. When complexed with both duplex and triplex, ethidium is characterized by hypochromism and a red shift in the absorption spectrum, a complicate induced circular dichroism (CD) band in the polynucleotide absorption region, and a negative reduced linear dichroism signal in both polynucleotide and drug absorption regions. The spectral properties for both duplex- and triplex-bound ethidium are identical and both can be understood by the intercalation binding mode. In contrast, the absorption and CD spectra of DAPI complexed with triplex differ from those of the DAPI-duplex complex, although both complexes can be understood by the intercalation binding mode. Considering that the third strand runs along the major groove of the template duplex, we conclude that the DAPI molecule partially intercalates near the major groove of the duplex, where the third strand can affect its spectroscopic properties.     

Base protonation facilitates B-Z interconversions of poly(dG-dC) X poly(dG-dC)

Comparative studies on the salt titration and the related kinetics for poly(dG-dC) X poly(dG-dC) in pH 7.0 and 3.8 solutions clearly suggest that base protonation facilitates the kinetics of B-Z interconversion although the midpoint for such a transition in acidic solution (2.0-2.1 M NaCl) is only slightly lower than that of neutral pH. The rates for the salt-induced B to Z and the reverse actinomycin D induced Z to B transitions in pH 3.8 solutions are at least 1 order of magnitude faster than the corresponding pH 7.0 counterparts. The lowering of the B-Z transition barrier is most likely the consequence of duplex destabilization due to protonation as indicated by a striking decrease (approximately 40 degrees C) in melting temperature upon H+ binding in low salt. The thermal denaturation curve for poly(dG-dC) X poly(dG-dC) in a pH 3.8, 2.6 M NaCl solution indicates an extremely cooperative melting at 60.5 degrees C for protonated Z DNA, which is immediately followed by aggregate formation and subsequent hydrolysis to nucleotides at higher temperatures. The corresponding protonated B-form poly(dG-dC) X poly(dG-dC) in 1 M NaCl solution exhibits a melting temperature about 15 degrees C higher, suggesting further duplex destabilization upon Z formation.     

Destabilization and stabilization of poly(A) · poly(U) structure by platinum(II) compounds

A methodology for analyzing the intramolecular structural order of the polynucleotide duplex poly(A) · poly(U) has been developed on the basis of molecular biophysics. The combination of circular dichroism spectroscopy and differential scanning calorimetry was shown to be an optimal approach. It ensures the screening of a wide set of substances and interaction conditions and the choice of compound(s) capable of stabilizing the structure and increasing the biological activity of this duplex. The study is aimed at obtaining a new and highly active antiviral drug.     

Binding of novel 9-O–N-aryl/arylalkyl amino carbonyl methyl berberine analogs to poly(U)-poly(A)·poly(U) triplex and comparison to the duplex poly(A)-poly(U)

Interaction of the 9-O–N-aryl/arylalkyl amino carbonyl methyl substituted analogs of the anticancer isoquinoline alkaloid berberine with RNA triplex, poly(U)-poly(A)·poly(U) has been studied in comparison to the duplex poly(A)-poly(U), using multiple biophysical techniques. Spectrophotometric and spectrofluorimetric studies established the non-cooperative binding mode of all the analogs with both the duplex and the triplex. However, berberine exhibited cooperative binding with poly(A)-poly(U) and non-cooperative binding with poly(U)-poly(A)·poly(U). Analog BER1 showed the highest affinity to both the duplex and the triplex followed by BER2 and BER3. The overall binding affinity varied as BER1 > BER2 > BER3 > BER. The magnitude of the quantum efficiency values (Q > 1) revealed that energy was transferred from the bases of the triplex and the duplex to the analogs. Comparative ferrocyanide quenching and viscosity studies unambiguously established a stronger intercalative geometry of the analogs to both the triplex and the duplex in comparison to berberine. Circular dichroism studies revealed that the alkaloids perturbed the conformation of both RNA helices. The binding of all the alkaloids was found to be exothermic from isothermal titration studies. Binding of the analogs was highly entropy driven while that of berberine was enthalpy dominated. The results presented here reveal strong and specific binding of these new berberine analogs to the RNA triplex and duplex and highlight the remarkable influence of the 9-substitution on the interaction profile.     

High pressure fourier transform infrared spectroscopy of poly(dA)poly(dT), poly(dA) and poly(dT)

The effect of hydrostatic pressure upon the DNA duplex, poly(dA)poly(dT), and its component single strands, poly(dA) and poly(dT) has been studied by fourier-transform infrared spectroscopy (FT-IR). The spectral data indicate that at 28 degrees C and pressures up to 12 kbar (1200 MPa) all three polymers retain the B conformation. Pressure causes the band at 967 cm(-1), arising from water-deoxyribose interactions, to shift to higher frequencies, a result consistent with increased hydration at elevated pressures. A larger pressure-induced frequency shift in this band is observed in the single stranded polymers than in the double stranded molecule, suggesting that the effect of pressure on the hydration of single strands may be greater than upon a double stranded complex. A pressure-dependent hypochromicity in the bands attributed to base stacking indicates that pressure facilitates the base stacking in the three polymers, in agreement with previous assessments of the importance of stacking in the stabilization of DNA secondary structure at ambient and high pressures.     

Antiviral activity of poly(A)-poly(U) duplexes modified with cis-diammine-dichloroplatinum (II)]

Polyribonucleotide duplex poly(A).poly(U) was modified with cis-diammine dichloroplatinum (II) (cis-DDP). It was shown that the antiinfluenza protective activity of the modified duplex in mice increased with the degree of modification (rb) rising up to 0.2. The effect was different from that for poly(I).poly(C) and poly(G).poly(C). The interferon titers in the murine brain increased in parallel with increasing of the antiviral activity. It was assumed that the structural specificity of the poly(A).poly(U) duplex was responsible for the phenomenon and that cis-DDP interaction with N(7) atoms of the adenine heterocycles blocked the "abnormal" Hoogsteen pairing of adenines with uracils. As a result the antiviral activity increased because of lowering the quantity of the intramolecular defects and increasing the length of the regular double-stranded regions.     

Interferon induction by platinum(II)-poly(I).poly(C) complexes

The effect of the interaction between poly(I).poly(C) and cis-dichloro-diammineplatinum(II) (cis-Pt), its trans analogue and chloro-diethylene-triamminoplatinum(II) (dien-Pt) on interferon induction activity was investigated. The covalent monodentate fixation of the three compounds on N7 of inosine has different effects on the structure and thermostability of poly(I). poly(C) which is well reflected by the interferon induction activity of the samples. Thus, the sandwich stabilization by dien-Pt at low binding ratios is manifested by an increased interferon induction and a high resistance towards RNAase degradation. The destabilization of the duplex by cis-Pt decreases interferon induction, accompanied by an increase in RNAase sensitivity of the complexes. In the case of trans-Pt the duplex structure is little perturbed and interferon induction is essentially maintained.     

Inhibition of poly(ADP-ribose)polymerase binding to DNA by thymidine dimer

The ability of poly(ADP-ribose)polymerase to bind damaged DNA was assessed by electrophoretic mobility shift assay. DNA binding domain of poly(ADP-ribose)polymerase (PARPDBD) binds to synthetic deoxyribonucleotide duplex 10-mer. However, the synthetic deoxyribonucleotide duplex containing cys-syn thymidine dimer which produces the unwinding of DNA helix structure lost its affinity to PARPDBD. It was shown that the binding of PARPDBD to the synthetic deoxyribonucleotide duplex was not affected by O6-Me-dG which causes only minor distortion of DNA helix structure. This study suggests that the stabilized DNA helix structure is important for poly(ADP-ribose)polymerase binding to DNA breaks, which are known to stimulate catalytic activity of poly(ADP-ribose)polymerase.     

Proton exchange and base-pair kinetics of poly(rA).poly(rU) and poly(rI).poly(rC)

Proton exchange of poly(rA).poly(rU) and poly(rI).poly(rC) has been studied by nuclear magnetic resonance line broadening and saturation transfer from H2O. Five exchangeable peaks are observed. They are assigned to the imino, amino and 2'-OH ribose protons. The aromatic spectrum is also assigned. Contrary to previous observations, we find that the exchange of the imino proton is strongly buffer sensitive. This property is used to derive the base-pair lifetime, which is in the range of milliseconds at 27 degrees C, 100 times smaller than published values. The enthalpy for the base-opening reaction (-86 kJ/mol) and the insensitivity of the reaction to magnesium suggest that the open state involves a small number of base-pairs. The similarities in the exchange from the two duplexes indicate that the same open state is responsible for exchange of purine and pyrimidine imino protons. For the lifetime of the open state and for the base-pair dissociation constant, we obtain only lower limits. At 27 degrees C they are three microseconds and 10(-3), respectively. The analysis that yields the much larger values published previously is based on the assumption that amino protons exchange only from open base-pairs. But theory and preliminary experiments indicate that it may occur from the closed duplex. The exchange of amino protons is slower than that of the imino protons. Exchange of the 2'-OH protons from the duplexes is much slower than from single-stranded poly(rU), and it is accelerated by magnesium. This could indicate hydrogen-bonding to backbone phosphate. Discrepancies between our results and those of previous studies are discussed.     

Structure of the beta-form of poly d(A).poly d(U)

The crystalline beta-form of the sodium salt of poly d(A).poly d(U) trapped in oriented fibers forms a Watson-Crick base-paired, 10(1) double-helix of pitch 3.2 nm. Two molecules are present in a monoclinic unit cell apparently isomorphous with beta-poly d(A).poly d(T). The two chains in each molecule both carry C2'-endo puckered furanose rings but are conformationally not identical. The orientations of the A:U base-pairs relative to the helix-axis are distinctly different from those in classical B-DNA and the overall morphology of the duplex in which they reside resembles that of the alpha-forms of poly (purine).poly (pyrimidine) DNA duplexes previously reported.     

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA)

To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.     

Binding of Hg(II) to poly(dA): poly(dT) and its component single strands

Mercuric binding studies at pH 10 revealed that poly(dA): poly(dT) exhibits a more dramatic absorption spectral alteration than the alternating polymer poly(dA-dT):poly(dA-dT) and induces a unique intense positive CD band at 296 nm during the spectral titrations. Comparative studies with its component single strands suggest that the spectral alterations exhibited by poly(dA): poly(dT) are consistent with a binding model in which the mercuric ions initially bind to thymines and cause the eventual strand separation of the duplex, with subsequent high cooperative binding to the poly(dA) strands. This interpretation is supported by the binding isotherms indicating much stronger mercuric binding to poly(dT) than to poly(dA), with saturation binding densities of 1 Hg(II) per 2 bases and 1 Hg(II) per base, respectively, and very high binding cooperativity for poly(dA). Striking spectral alterations are exhibited by the mercuric binding to poly(dA), likely the consequence of binding to the amino group of dA in an alkaline solution. The mononucleoside dA exhibits minor spectral alterations upon similar mercuric chloride additions whereas the dinucleoside monophosphate d(AA) exhibits significant spectral changes, albeit less pronounced than those of poly(dA). Some sequence effects on the mercuric binding are observed in the dinucleotide studies. Our CD results on the mercuric binding to polynucleotides do not support the contention of (psi)-type condensed complex formation.     

Infrared and Raman studies show that poly(dA).poly(dT) and d(AAAAATTTTT)2 exhibit a heteronomous conformation in films at 75% relative humidity and a B-type conformation at high humidities and in solution

The decadeoxynucleotide d(AAAAATTTTT)2 in duplex form and the double-helical polynucleotide poly(dA).poly(dT) have been studied by Raman and infrared (IR) spectroscopy under a variety of environmental conditions. The IR spectra have been taken of cast films and compared to the IR spectra of the alternating poly(dA-dT), which shows clear B-genus and A-genus vibrational spectra under conditions of high (greater than 92%) and low (75%) relative humidity (RH). From the IR data, it is shown that d-(AAAAATTTTT)2 and poly(dA).poly(dT) adopt a B-genus conformation in films with high water content. When the relative humidity of the film is decreased, the IR spectra reflect a gradual evolution of the geometry of both d(AAAAATTTTT)2 and poly(dA).poly(dT) into a form intermediate between the B genus and A genus, but the IR spectrum of a pure A genus has not been obtained. In these DNAs at 75% RH, the IR bands of adenosine have the same frequencies as those found in poly(dA-dT) at 75% RH where the local furanose conformation is C3' endo/anti, but the thymidine frequencies do not resemble those of poly(dA-dT) at 75% RH but rather those of poly(dA-dT) at high humidities. It is concluded that both poly(dA).poly(dT) and d(AAAAATTTTT)2 adopt a fully heteronomous duplex geometry in cast films at low humidity. For studies in aqueous solution the Raman effect was employed. As a model for the heteronomous conformation in solution, the duplex poly(rA).poly(dT) was used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of a pleiomeric form of poly d(AT):poly d(AT)

A chemically simple polynucleotide duplex, poly d(AT):poly d(AT), has been trapped in a fibrous form with a complex helical secondary structure with a large (7.4 nm) axial repeat 24 nucleotides long. The motif which is repeated by the symmetry elements is a hexanucleotide in which two residues (both TpA) have the less common gauche minus conformation at C3'-O3' and consequently distinctive phosphate orientations. This reinforces earlier conclusions that PypPu nucleotides tend to have different shapes from PupPy nucleotides and that DNA surfaces may signal what base sequences lie beneath them. The morphological differences between this pleiomeric DNA polymer and closely-related, but more symmetrical allomorphs are just as great as those observed in short DNA fragments in crystals.     

Interferon induction by platinum(II)-poly(I)·poly(C) complexes

The effect of the interaction between poly(I)·poly(C) and cis-dichloro-diammineplatinum(II) (cis-Pt), its trans analogue and chloro-diethylene-triamminoplatinum(II) (dien-Pt) on interferon induction activity was investigated. The covalent monodentate fixation of the three compounds on N⁷ of inosine has different effects on the structure and thermostability of poly(I)·poly(C) which is well reflected by the interferon induction activity of the samples. Thus, the sandwich stabilization by dien-Pt at low binding ratios is manifested by an increased interferon induction and a high resistance towards RNAase degradation. The destabilization of the duplex by cis-Pt decreases interferon induction, accompanied by an increase in RNAase sensitivity of the complexes. In the case of trans-Pt the duplex structure is little perturbed and interferon induction is essentially maintained.     

A monoclonal antibody specific for the duplex DNA poly[d(TC)].poly[d(GA)]

Although most duplex DNAs are not immunogenic some synthetic DNAs such as poly[d(Tm5C)].poly[d(GA)] are weakly immunogenic allowing the production of monoclonal antibodies. The specificity of one of these antibodies, Jel 172, was investigated in detail by a competitive solid-phase radioimmune assay. Jel 172 bound well to poly[d(TC)].poly[d(GA)] but not to other duplex DNAs such as poly[d(TTC)].poly[d(GAA)] and poly[d(TCC)].poly[d(GGA)]. The binding to poly[d(Br5UC)].poly[d(GA)] was enhanced while that to poly[d(TC)].poly[d(IA)] was decreased compared to poly[d(TC)].poly[D(GA)]. Thus, not only is the antibody very specific for a sequence of duplex DNA but it also appears to recognize functional groups in both grooves of the helix.     

The binding of poly(rI) - poly(rC) to human fibroblasts and the induction of interferon.

Human embryonic fibroblasts produce interferon when incubated at 37 degrees C after being treated at 4 degrees C with poly(rI) - poly(rC), either by addition of the double-stranded duplex or by sequential addition of the constitutent single-stranded polynucleotides. Cells which have been incubated with double-stranded poly(rI) - poly(rC) can be prevented from forming interferon by washing the cells with high concentrations of salt, immediately after adsorption of polynucleotides, or by incubation of the cells with single-stranded polynucleotides. The inhibition is probably due to displacement of the inducing molecule from the cell surface. Interferon production by cells treated sequentially with poly(rI) and poly(rC) is not inhibited by either of these treatments and the polynucleotides are not easily displaced from the cell surface.