A Raman spectroscopic study of the interaction between nucleotides and the DNA binding protein gp32 of bacteriophage T4

Raman spectra of the bacteriophage T4 denaturing protein gp32, its complex with the polynucleotides poly(rA), poly(dA), poly(dT), poly(rU), and poly(rC), and with the oligonucleotides (dA)₈ and (dA)₂, were recorded and interpreted. According to an analysis of the gp32 spectra with the reference intensity profiles of Alix and co-workers [M. Berjot, L. Marx, and A. J. P. Alix (1985) J. Ramanspectrosc., submitted; A. J. P. Alix, M. Berjot, and J. Marx (1985) in Spectroscopy of Biological Molecules, A. J. P. Alix, L. Bernard, and M. Manfait, Eds., pp. 149–154], 1 gp32 contains ≈ 45% helix, ≈ 40% β-sheet, and 15% undefined structure. Aggregation of gp32 at concentrations higher than 40 mg/mL leads to a coordination of the phenolic OH groups of 4–6 tyrosines and of all the sulfhydryl (SH) groups present in the protein with the COO^? groups of protein. The latter coordination persists even at concentrations as low as 1 mg/mL. In polynucleotide–protein complexes the nucleotide shields the 4–6 tyrosine residues from coordination by the COO^? groups even at high protein concentration. The presence of the nucleotide causes no shielding of the SH groups. With Raman difference spectroscopy it is shown that binding of the protein to a single-stranded nucleotide involves both tyrosine and trytophan residues. A change in the secondary structure of the protein upon binding is observed. In the complex, gp32 contains more β-sheet structure than when uncomplexed. A comparison of the spectra of complexed poly(rA) and poly(dA) with the spectra of their solution conformations at 15°C reveals that in both polynucleotides the phosphodiester vibration changes upon complex formation in the same way as upon a transition from a regular to a more disordered conformation. Distortion of the phosphate–sugar–base conformation occurs upon complex formation, so that the spectra of poly(rA) and poly(dA) are more alike in the complex than they are in the free polynucleotides. The decrease in intensity of the Raman bands at 1304 cm^?1 in poly(rA), at 1230 cm^?1 in poly(rU), and at 1240 and 1378 cm^?1 of poly(dT) may be indicative of increased stacking interactions in the complex. No influence of the nucleotide chain length upon the Raman spectrum of gp32² in the complex was detected. Both the nucleotide lines and the protein lines in the spectrum of a complex are identical in poly(dA) and (dA)₈.     

Resonance Raman spectroscopy of complexes of the helix destabilizing proteins GP32 and GP5 with poly(rA) and poly(dA)

The bacteriophage T4 helix destabilizing protein (hdp) gp32 and its complexes with poly(rA) and poly(dA) were studied with ultra-violet resonant Raman spectroscopy. The UV-resonant Raman (UV-RR) spectrum of the complex of gp5, the coat protein of bacteriophage M13, with poly(dA) was also measured and is compared with the spectrum of the gp 32/poly(dA) complex. The excitation wavelength was 245.1 nm. This is on the far UV-side of the first absorption bands of adenine and near a "window" in the protein absorption spectrum. The overlap of fluorescence due to chromophores present in the protein and resonance Raman scattering was prevented by this choice of wavelength. The spectra of the protein/polynucleotide complexes are compared with the native nucleotide spectra measured at varying temperatures. The hyperchromicity which is expected when a nucleotide changes from a stacked to an unstacked conformation was not observed for poly(rA), neither upon temperature increase nor on protein binding. In both cases poly(dA) revealed a clear hyperchromicity. This different behavior of poly(rA) and poly(dA) is probably a consequence of their different conformations. The contributions of the proteins to the spectra is weak except for two bands, at 1550 and 1610 cm-1 due to tryptophan (in case of gp32) and one band near 1610 cm-1 due to tyrosine and phenylalanine.     

Binding of CC-1065 to poly- and oligonucleotides

The binding of the antitumor agent CC-1065 to a variety of poly- and oligonucleotides was studied by electronic absorption, CD, and resistance to removal by Sephadex column chromatography. Competitive binding experiments between CC-1065 and netropsin were carried out with calf-thymus DNA, poly(dI-dC) · poly(dI-dC), poly(dI) · poly(dC), poly(rA) · poly(dT), poly(dA- dC) · poly(dG-dT), and poly(dA) · 2poly(dT). CC-1065 binds to polynucleotides by three mechanisms. In the first, CC-1065 binds only weakly, as judged by the induction of zero or very weak CD spectra and low resistance to extraction of drug from the polynucleotide by Sephadex chromatography. In the second and third mechanisms, CC-1065 binds strongly, as judged by the induction of two distinct, intense CD spectra and high resistance to extraction of drug from the polynucleotide, by Sephadex chromatography in both cases. The species bound by the second mechanism converts to that bound by the third mechanism with varying kinetics, which depend both on the base-pair sequence and composition of the polynucleotide. Competitive binding experiments with netropsin show that CC-1065 binds strongly in the minor groove of DNA by the second and third mechanisms of binding. Netropsin can displace CC-1065 that is bound by the second mechanism but not that bound by the third mechanism. CC-1065 binds preferentially to B-form duplex DNA and weakly (by the first binding mechanism) or not at all to RNA, DNA, and RNA–DNA polynucleotides which adopt the A-form conformation or to single-strand DNA. This correlation of strong binding of CC-1065 to B-form duplex DNA is consistent with x-ray data, which suggest an anomalous structure for poly(dI) · poly(rC), as compared with poly(rI) · poly(dC) (A-form) and poly(dI) · poly(dC) (B-form). The binding data indicate that poly(rA) · poly(dU) takes the B-form secondary structure like poly(rA) · poly(dT). Triple-stranded poly(dA) · 2poly(dT) and poly(dA) · 2poly(dU), which are considered to adopt the A-form conformation, bind CC-1065 strongly. Netropsin, which also shows a binding preference for B-form polynucleotides, also binds to poly(dA) · 2poly(dT) and occupies the same binding site as CC-1065. These binding studies are consistent with results of x-ray studies, which suggest that A-form triplex DNA retains some structural features of B-form DNA that are not present in A-form duplex DNA; i.e., the axial rise per nucleotide and the base tilt. Triple-stranded poly(dA) · 2poly(rU) does not bind CC-1065 strongly but has nearly the same conformation as poly(dA) · 2poly(dT) based on x-ray analysis. This suggests that the 2′-OH group of the poly(rU) strands interferes with CC-1065 binding to this polynucleotide. The same type of interference may occur for other RNA and DNA–RNA polynucleotides that bind CC-1065 weakly.     

The poly dA strand of poly dA.poly dT adopts an A-form in solution: a UV resonance Raman study.

The study by resonance Raman spectroscopy with a 257 nm excitation wave-length of adenine in two single-stranded polynucleotides, poly rA and poly dA, and in three double-stranded polynucleotides, poly dA.poly dT, poly(dA-dT).poly(dA-dT) and poly rA.poly rU, allows one to characterize the A-genus conformation of polynucleotides containing adenine and thymine bases. The characteristic spectrum of the A-form of the adenine strand is observed, except small differences, for poly rA, poly rA.poly rU and poly dA.poly dT. Our results prove that it is the adenine strand which adopts the A-family conformation in poly dA.poly dT.     

Study of the binding of single-stranded DNA-binding protein to DNA and poly(rA) using electric field induced birefringence and circular dichroism spectroscopy

Binding of the single-stranded DNA-binding protein (SSB) of Escherichia coli to single-stranded (ss) polynucleotides produces characteristic changes in the absorbance (OD) and circular dichroism (CD) spectra of the polynucleotides. By use of these techniques, complexes of SSB protein and poly(rA) were shown to display two of the binding modes reported by Lohman and Overman [Lohman, T.M., & Overman, L. (1985) J. Biol. Chem. 260, 3594-3603]. The circular dichroism spectra of the "low salt" (10 mM NaCl) and "high salt" (greater than 50 mM NaCl) binding mode are similar in shape, but not in intensity. SSB binding to poly(rA) yields a complexed CD spectrum that shares several characteristics with the spectra obtained for the binding of AdDBP, GP32, and gene V protein to poly(rA). We therefore propose that the local structure of the SSB-poly(rA) complex is comparable to the structures proposed for the complexes of these three-stranded DNA-binding proteins with DNA (and RNA) and independent of the SSB-binding mode. Electric field induced birefringence experiments were used to show that the projected base-base distance of the complex is about 0.23 nm, in agreement with electron microscopy results. Nevertheless, the local distance between the successive bases in the complex will be quite large, due to the coiling of the DNA around the SSB tetramer, thus partly explaining the observed CD changes induced upon complexation with single-stranded DNA and RNA.     

The Conformation of the Complex of the Helix Destabilizing Protein GP32 of Bacteriophage T4 and Single Stranded DNA

Abstract

The conformation of single stranded polynucleotides is changed specifically upon binding of the helix destabilizing protein of bacteriophage T4 (GP32). On the basis of circular dichroism (CD) and absorption experiments it is shown that denaturing conditions and the binding of oligopeptides can not induce the altered conformation. On the contrary, according to the current CD and absorption theory, the optical properties of the complex can be explained by a specific, regular conformation, characterized by an appreciable tilt of the bases (?—10°) and either a small rotation per base or a small helix diameter. This conformation agrees nicely with the increase of the base-base distance in the complex as determined in solution by electric field induced birefringence measurements. Our calculations show that also the model proposed by Alma (Ph.D. Thesis Catholic University Nijmegen, The Netherlands (1982)) for the complex of the helix destabilizing protein of bacteriophage fd, in which the helix diameter is large and the bases are almost parallel to the helix axis, would agree with the CD- and absorption spectra of the GP32-complex. For the latter protein this model would have to be modified with regard to the axial increment of the bases which is much larger in the GP32-complexes.     

The conformation of the complex of the helix destabilizing protein GP32 of bacteriophage T4 and single stranded DNA

The conformation of single stranded polynucleotides is changed specifically upon binding of the helix destabilizing protein of bacteriophage T4 (GP32). On the basis of circular dichroism (CD) and absorption experiments it is shown that denaturing conditions and the binding of oligopeptides can not induce the altered conformation. On the contrary, according to the current CD and absorption theory, the optical properties of the complex can be explained by a specific, regular conformation, characterized by an appreciable tilt of the bases (less than or equal to -10 degrees) and either a small rotation per base or a small helix diameter. This conformation agrees nicely with the increase of the base-base distance in the complex as determined in solution by electric field induced birefringence measurements. Our calculations show that also the model proposed by Alma (Ph.D. Thesis Catholic University Nijmegen, The Netherlands (1982)) for the complex of the helix destabilizing protein of bacteriophage fd, in which the helix diameter is large and the bases are almost parallel to the helix axis, would agree with the CD- and absorption spectra of the GP32-complex. For the latter protein this model would have to be modified with regard to the axial increment of the bases which is much larger in the GP32-complexes.     

Triple helical polynucleotidic structures: sugar conformations determined by FTIR spectroscopy.

Fourier Transform Infrared Spectra of triple stranded polynucleotides containing homopurine dA or rA and homopyrimidine dT or rU strands have been obtained in H2O and D2O solutions as well as in hydrated films at various relative humidities. The spectra are interpreted by comparison with those of double stranded helixes with identical base and sugar composition. The study of the spectral domain corresponding to in-plane double bond stretching vibrations of the bases shows that whatever the initial duplex characterized by a different IR spectrum (A family form poly rA.poly rU, heternomous form poly rA.poly dT, B family form poly dA.poly dT), the triplexes present a similar IR spectrum reflecting similar base interactions. A particular attention is devoted to the 950-800 cm-1 region which contains marker bands of the sugar conformation in the nucleic acids. In solution the existence of only N (C3'endo-A family form) type of sugar pucker is detected in poly rU.poly rA.poly rU and poly dt.poly rA.poly rU. On the contrary absorption bands characteristic of both N (C3'endo-A family form) and S (C2'endo-B family form) type sugars are detected for poly rU.poly rA.poly dT, poly rU.poly dA.poly dT and poly dT.poly rA.poly dT. Finally mainly S (C2'endo-B family form) type sugars are observed in poly dT.poly dA.poly dT.     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA).poly(dT) and poly(dG).poly(dC), and with triple helical poly(dA).[poly(dT)](2) and poly(dC).poly(dG).poly(dC)(+) were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA).poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG).poly(dC) and -poly(dC).poly(dG).poly(dC)(+) complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

A Raman scattering study of the helix-destabilizing gene-5 protein with adenine-containing nucleotides.

Raman spectra of gp5 and complexes of gp5 with poly(rA) and poly(dA) have been determined and analysed. From a fit of the amide I-band with model spectra it follows that the secondary structure of gp5 contains 52% beta-sheet, 28% undefined conformation and 19% alpha-helix. The band at 1032 cm-1 due to phenylalanine has an anomalous intensity both in the spectra of the complexes and the free protein. This possibly indicates a stacked structure present in the protein. Binding of gp5 to poly(rA) and poly(dA) influences the intensity of bands near 1338 and 1480 cm-1 which are considered to be marker-bands for the phosphate-sugar-base conformer. A change in conformation of the nucleotides is also reflected by vibrations originating in the phosphate- and sugar-residues of the backbone. In the spectrum of complexed poly(rA) the intensity of the conformation sensitive band at 813 cm-1, which is due to the phosphodiester group, is zero. It seems that gp5 forces poly(rA) and poly(dA) to a similar conformation. A marker band for stacking interaction in poly(rA) indicates that stacking interactions in the complex have increased.     

Binding of Mg2+ to single-stranded polynucleotides: hydration and optical studies

The binding of Mg(2+) to single-stranded ribo- and deoxy-polynucleotides, poly(rA), poly(rU), poly(dA) and poly(dT), has been investigated in dilute aqueous solutions at pH 7.5 and 20 degrees C. A combination of ultrasound velocimetry, density, UV and CD spectroscopy have been employed to study hydration and spectral effects of Mg(2+) binding to the polynucleotides. Volume and compressibility effects of Mg(2+) binding to random-coiled poly(rU) and poly(dT) correspond to two coordination bonds probably between the adjacent phosphate groups. The same parameters for poly(rA)+Mg(2+) correspond to an inner-sphere complex with three-four direct contacts. However, almost no hydration effects are arising in binding to its deoxy analog, poly(dA), indicating mostly a delocalized binding mode. In agreement with hydration studies, optical investigations revealed almost no influence of Mg(2+) on poly(dA) properties, while it stabilizes and aggregates poly(rA) single-helix. The evidence presented here indicates that Mg(2+) are able to bind specifically to single-stranded polynucleotides, and recognize their composition and backbone conformation.     

Orientation of the bases of single-stranded DNA and polynucleotides in complexes formed with the gene 32 protein of bacteriophage T4. A linear dichroism study

Linear dichroism measurements were performed in the wavelength region 250 to 350 nm on complexes between the single-stranded DNA binding protein of bacteriophage T4 (gp32) and single-stranded DNA and a variety of homopolynucleotides in compressed polyacrylamide gels. The complexes appeared to orient well, giving rise to linear dichroism spectra that showed contributions from both the protein aromatic residues and the bases of the polynucleotides. In most cases the protein contribution appeared to be very similar, and the linear dichroism of the bases could be explained by similar orientations of the bases for most of the complexes. Assuming a similar, regular structure for most of the polynucleotides in complex, only a limited set of combinations of tilt and twist angles can explain the linear dichroism spectra. These values of tilt and twist are close to (-40 degrees, 30 degrees), (-40 degrees, 150 degrees), (40 degrees, -30 degrees) or (40 degrees, -150 degrees), with an uncertainty in both angles of about 15 degrees. Although the linear dichroism results do not allow a choice between these possible orientations, the latter two combinations are not in agreement with earlier circular dichroism calculations. For the complexes formed with poly(rC) and poly(rA), the linear dichroism spectra could not be explained by the same base orientations. In these two cases also the protein contribution to the linear dichroism appeared to be different, indicating that for some aromatic residues the orientations are not the same as those in the other complexes. The different structures of these complexes are possibly related to the relatively low binding affinity of gp32 to poly(rC), and to a lesser extent to poly(rA).     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA)·poly(dT) and poly(dG)·poly(dC), and with triple helical poly(dA)·[poly(dT)]₂ and poly(dC)·poly(dG)·poly(dC)⁺ were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA)·poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG)·poly(dC) and -poly(dC)·poly(dG)·poly(dC)⁺ complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

Investigation of complexes formed between gene 32 protein from bacteriophage T4 and heavy-atom-modified single-stranded polynucleotides using optical detection of magnetic resonance

Optical detection of triplet-state magnetic resonance (ODMR) is employed to study the complexes formed between gene 32 protein (GP32), a single-stranded DNA-binding protein from bacteriophage T4, and the heavy-atom-derivatized polynucleotides poly(5-HgU) and poly(5-BrU). The triplet-state properties of some of the tryptophan (Trp) residues in the complexes are dramatically different from those in the free protein, in that they are subject to an external heavy-atom effect. Direct evidence for the presence of a heavy-atom effect, and hence a close-range interaction between mercurated or brominated nucleotide bases and Trp residues in the complex, is provided by the observation of the zero-field (D) + (E) ODMR transition of Trp, which is not normally observed in the absence of a heavy-atom perturbation. The amplitude-modulated phosphorescence-microwave double-resonance (AM-PMDR) technique is employed to selectively capture the phosphorescence spectrum originating from the heavy-atom-perturbed Trp residue(s) in the GP32-poly(5-HgU) complex. Arguments based on our experimental results lead to the conclusion that the heavy-atom perturbation arises from aromatic stacking interactions between Trp and mercurated bases. Wavelength-selected ODMR measurements reveal the existence of two environmentally distinct and spectrally different types of Trp in GP32. One of these types is perturbed selectively by the heavy atom and hence undergoes stacking interactions with the heavy-atom-derivatized bases of the polynucleotide while the second type of Trp residue is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polynucleotide sequences in eukaryotic DNA and RNA that form ribonuclease-resistant complexes with polyuridylic acid

When annealed with synthetic polynucleotides and treated with ribonuclease under appropriate conditions, poly(U) forms the ribonuclease-resistant complexes poly(rA) · poly(U) (1:1), poly(dA) · 2poly(U) (1:2) and poly · (dA)poly(dT) · poly(U) (1:1:1). This forms the basis of a quantitative assay of poly(rA), poly(dA) and poly(dA) · poly(dT) sequences in unlabelled nucleic acids. Using this assay, duck haemoglobin messenger RNA is shown to contain a poly(rA) sequence approximately 100 nucleotides long.Eukaryotic DNAs contain small amounts of sequences that react with poly(U). In the case of duck DNA, these sequences are considerably shorter than the mRNA-associated sequences and are interspersed widely with other sequences. It is concluded that if duck DNA does contain poly(dA) sequences corresponding to mRNA-associated poly(rA) sequences, there are fewer than 8000 of these per haploid genome.     

Spectroscopic analysis of the interaction of Escherichia coli DNA-dependent RNA polymerase with T7 DNA and synthetic polynucleotides.

We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA.     

Raman studies of nucleic acids. XII. Conformations of oligonucleotides and deuterated polynucleotides

Laser Raman spectra of the trinucleoside diphoshate ApApA and dinucleoside phosphates ApU, UpA, GpC, CpG, and GpU are reported and discussed. Assignments of conformationally sensitive frequencies are-facilitated by comparison with spectra reported here of poly(rA), poly(rC), and poly(rU) in deuterium oxide solutions. The significant spectral differences between ApU and UpA, and between GpC and CpG, reveal that the sequence isomers have nonidentical conformations in aqueous solution. In UpA at low temperature the bases are stacked and the backbone conformation is similar to that found in ordered polynucleotide structures and RNA. In ApU no base stacking can be detected and the backbone conformation differs from that found in UpA, both in the orientation of phosphodiester linkages and in the internal conformation of ribose. At the conditions employed neither ApU nor UpA exhibits base pairing in aqueous solutions. In both GpC and CpG the bases are stacked and the phosphodiester conformations are similar to those encountered for UpA and RNA. However, major differences between spectra of GpC and CpG indicate that the geometries of stacking and ribosyl conformations are different. In GpC the Raman data favor the formation of hydrogen bonded dimers containing GC pairs. Protonation of C in GpC is sufficient to eliminate the ordered conformation detected by Raman spectroscopy. Despite the ordered backbone conformation evident in GpU, this dinucleoside apparently contains neither stacked nor hydrogen bonded bases at the conditions employed here. The Raman data also confirm the stacking interactions in ApApA, poly(rA), and poly(rC) but suggest that the backbone conformation in poly(rC) differs qualitatively from that found in most ordered polynucleotide structures and is thermally more stable. The present results demonstrate the sensitivity of the Raman technique to sequence-related structural differences in oligonucleotides and provide additional spectra–structure correlations for future conformational studies of RNA by laser Raman spectroscopy.     

Interaction Specificity of Benzimidazol Group Compounds with AT-Containing Polynucleotides

Abstract

The interaction between polynucleotides: poly(dA)-poly(dT), poly(dA-dT), poly(am2dA- dT), and the AT-specific compounds of benzimidazol group has been studied. It is been shown that these compounds bind to poly(dA)-poly(dT) and poly(dA-dT) at low and high salt concentration in solution. Poly(am2dA-dT) interacts with AT-specific compounds only at low salt, where this polynucleotide is in a B-form, but not at high salt when the polynucleotide converts to another conformation. Thus, the interaction specificity of the groove-binding ligands is influenced not only by the minor groove substituents, but the peculiarities of the secondary structure of polynucleotides.     

Binding geometries of triple helix selective benzopyrido [4,3-b]indole ligands complexed with double- and triple-helical polynucleotides

The binding modes of three benzopyrido [4,3-b]indole derivatives (and one benzo[-f]pyrido [4-3b] quinoxaline derivative) with respect to double helical poly(dA) · poly(dT) and poly[d(A-T)]₂ and triple-helical poly(dA) · 2poly(dT) have been investigated using linear dichroism (LD) and CD: (I) 3-methoxy-11-amino-BePI where BePI = (7H-8-methyl-benzo[e]pyrido [4,3-b]indole), (II) 3-methoxy-11-[(3′-amino) propylamino]-BePI, (III) 3-methoxy-7-[(3′-diethylamino)propylamino] BgPI where BgPI = (benzo[g]pyrido[4,3-b]indole), and (IV) 3-methoxy-11-[(3′-amino)propylamino] B f P Q where B f P Q = {benzo[-f]pyrido[4-3b]quinoxaline}. The magnitudes of the reduced LD of the electronic transitions of the polynucleotide bases and of the bound ligands are generally very similar, suggesting an orientation of the plane of the ligands' fused-ring systems preferentially perpendicular to the helix axis. The LD results suggest that all of the ligands are intercalated for all three polynucleotides. The induced CD spectrum of the BePI chromophore in the (II-BePI)-poly[d(A-T)]₂ complex is almost a mirror image of that for the (I-BePI)-poly(dA) · poly(dT) and (I-BePI)-poly(dA) · 2poly(dT) complexes, suggesting an antisymmetric orientation of the BePI moiety upon intercalation in poly[d(A-T)]₂ compared to the other polynucleotides. The induced CD of I-BePI bound to poly(dA) · 2poly(dT) suggests a geometry that is intermediate between that of its other two complexes. The concluded intercalative binding as well as the conformational variations between the different BePI complexes are of interest in relation to the fact that BePI derivatives are triplex stabilizers. © 1997 John Wiley & Sons, Inc. Biopoly 42: 101–111, 1997