A Theoretical Investigation on the Sequence Selective Binding of Daunomycin to Double-Stranded Polynucleotides

Abstract

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of daunomycin (DNM) to six representative self- complementary double-stranded hexanucleotides: d(CGTACG)₂, d(CGATCG)₂, d(CITACI)₂, d(TATATA)₂, d(CGCGCG)₂ and d(TACGTA)₂. The conformational angles of the hexanucleotides are fixed in values found in the representative crystal structure of the d(CGTACG)₂- DNM complex. The intermolecular DNM-hexanucleotide interaction energies and the conformational energy changes of DNM upon binding are computed and optimized in the framework of the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. Among the two regularly alternating hexanucleotides, d(TATATA)₂ and d(CGCGCG)₂, a stronger binding is predicted for the former, in agreement with experimental results obtained with poly(dA-dT)-poly(dA-dT) and poly(dG-dC)<<poly(dG-dC). Altogether, however, among the six investigated sequences, the strongest complexes are computed for the mixed hexanucleotides d(CGATCG)₂ and d(CGTACG)₂, containing the intercalation site between two CG base pairs and an adjacent TA base pair. This situation may be related to the increased affinity of DNM for GC rich DNA's and to the situation in the crystal structure of the DNM-d(CGTACG)₂ complex. Analysis of the intrinsic base sequence preferences expressed by the individual constituents of DNM, namely the daunosamine side chain, the chromophore ring and its two 9-hydroxyl and 9-acetoxy substituents, reveals that the overall sequence preference found is the result of a rather intricate interplay of intrinsic sequence preferences, in particular at the level of daunosamine and the 9-hydroxyl substituent. Altogether, it is seen that the selective base pair recognition by daunomycin cannot, in general, be defined in terms of the two base pairs implicated in the intercalation site alone (with the exception of homogeneous AT or GC base sequences) but must be expressed in terms of a triplet of base pairs.     

A theoretical investigation on the sequence selective binding of daunomycin to double-stranded polynucleotides

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of daunomycin (DNM) to six representative self-complementary double-stranded hexanucleotides: d(CGTACG)2,d(CGATCG)2,d(CITACI)2, d(TATATA)2, d(CGCGCG)2 and d(TACGTA)2. The conformational angles of the hexanucleotides are fixed in values found in the representative crystal structure of the d(CGTACG)2-DNM complex. The intermolecular DNM-hexanucleotide interaction energies and the conformational energy changes of DNM upon binding are computed and optimized in the framework of the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. Among the two regularly alternating hexanucleotides, d(TATATA)2 and d(CGCGCG)2, a stronger binding is predicted for the former, in agreement with experimental results obtained with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Altogether, however, among the six investigated sequences, the strongest complexes are computed for the mixed hexanucleotides d(CGATCG)2 and d(CGTACG)2, containing the intercalation site between two CG base pairs and an adjacent TA base pair. This situation may be related to the increased affinity of DNM for GC rich DNA's and to the situation in the crystal structure of the DNM-d(CGTACG)2 complex. Analysis of the intrinsic base sequence preferences expressed by the individual constituents of DNM, namely the daunosamine side chain, the chromophore ring and its two 9-hydroxyl and 9-acetoxy substituents, reveals that the overall sequence preference found is the result of a rather intricate interplay of intrinsic sequence preferences, in particular at the level of daunosamine and the 9-hydroxyl substituent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and hydropathic characterization of structural features affecting sequence specificity for doxorubicin intercalation into DNA double-stranded polynucleotides.

The computer molecular modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field function that includes hydrogen bonding, coulombic and hydrophobic terms, was used to study sequence-selective doxorubicin binding/intercalation in the 64 unique CAxy, CGxy, TAxy, TGxy base pair quartet combinations. The CAAT quartet sequence is shown to have the highest binding score of the 64 combinations. Of the two regularly alternating polynucleotides, d(CGCGCG)2and d(TATATA)2, the HINT calculated binding scores reveal doxorubicin binds preferentially to d(TATATA)2. Although interactions of the chromophore with the DNA base pairs defining the intercalation site [I-1] [I+1] and the neighboring [I+2] base pair are predominant, the results obtained with HINT indicate that the base pair [I+3] contributes significantly to the sequence selectivity of doxorubicin by providing an additional hydrogen bonding opportunity for the N3' ammonium of the daunosamine sugar moiety in approximately 25% of the sequences. This observation, that interactions involving a base pair [I+3] distal to the intercalation site play a significant role in stabilizing/destabilizing the intercalation of doxorubicin into the various DNA sequences, has not been previously reported. In general terms, this work shows that molecular modeling and careful analysis of molecular interactions can have a significant role in designing and evaluating nucleotides and antineoplastic agents.     

Association of anthracyclines and synthetic hexanucleotides. Structural factors influencing sequence specificity

The equilibrium and kinetic aspects of the interaction between four anthracyclines and two synthetic self-complementary hexanucleotides was investigated by fluorescence detection. Two of the studied anthracyclines are widely used antitumor drugs: doxorubicin (1, formerly adriamycin) and daunorubicin (2, formerly daunomycin). The other two, 9-deoxydoxorubicin (3) and 3'-deamino-3'-hydroxy-4'-epidoxorubicin (4), are doxorubicin analogues with modifications of the chemical groups that have been proposed as responsible for sequence specificity (Chen, K.-X., Gresh, N. and Pullman, B. (1985). J. Biomol. Struct. Dyn. 3, 445-466). One of the oligonucleotides, d(CGTACG), is identical to that used in the high resolution x-ray structure determination of the daunorubicin intercalative complex (Wang, A. H.-J., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Biochemistry 26, 1152-1163). Binding to this hexanucleotide is compared with intercalation into the d(CGCGCG) duplex, revealing sequence preferences of the four anthracyclines. Taking into account the anthracycline aggregation and the dissociation of the hexanucleotide double standard form, results can be interpreted with a model that assumes complete fluorescence quenching at intercalative sites containing the CG base pair, and a large residual fluorescence after intercalation within the TpA fragment. All four anthracyclines show preferential intercalation at sites near the ends of both hexanucleotide duplexes, partly as a result of positive cooperativity in the formation of di-intercalated species at these sites. Within the limits of experimental error, complete site specificity for the CpG fragment is found in the intercalation of 1 and 2 into d(CGTACG) duplex, whereas analogues 3 and 4 give increasing evidence of intercalation at other sites including the fluorescence-preserving TpA fragment. Site specificity is less pronounced in the association with d(CGCGCG), when cooperativity is taken into account. Kinetic data corroborate the results of equilibrium studies and are interpreted with a mechanism that includes formation of an intermediate bound species followed by drug redistribution to preferential sites. Finally, from a comparison of pertinent site binding constants, approximate free energy contributions to sequence specific DNA interaction, due to C9-OH on the aglycone and -NH3+ on daunosamine, are estimated not to exceed 2 kcal/mol.     

Modelling basic features of specificity in the binding of a dicationic steroid diamine to double-stranded oligonucleotides.

An investigation of the intrinsically preferred binding modes of a steroid diamine, dipyrandium, to the double-stranded hexanucleotides d(TATATA)2, d(ATATAT)2, and d(CGCGCG)2 is carried out by the energy minimization procedure JUMNA. Several alternative binding modes are compared: groove binding in which the conformation of the oligonucleotide remains close to that of B-DNA, intercalation between base-pairs and interaction with variously kinked structures in which base pairs of dinucleoside steps open towards the groove in which the binding occurs. The favored binding configuration occurs at the d(TpA) step of the AT kinked nucleotides in which the kink opens the base pairs towards the minor groove. Thus, for the d(T1A2T3A4T5A6)2 sequences the preferred complexation involves the kink at the T3A4 step facing the cyclohexane rings A, B, and C of the ligand. For the d(A1T2A3T4A5T6)2 sequence, the kink occurs at the T2A3 step facing the cationic pyrrolidine ring linked to ring A. The binding of dipyrandium to d(CGCGCG)2 is found to be considerably less favourable than for either of the two (AT) sequences.     

On the sequence selective bis-intercalation of a homodimeric thiazole orange dye in DNA.

The thiazole orange dye 1,1'-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-[(3-methyl-2,3-dihydro(benzo-1, 3-thiazolyl)-2-methylidene]quinolinium tetraiodide (TOTO) binds sequence selectively to double-stranded DNA (dsDNA) by bis-intercalation. Each chromophore is sandwiched between two base pairs in a d(5'-py-p-py-3'):d(5'-pu-p-pu-3') site, and the linker spans over two base pairs in the minor groove. We have examined the binding of TOTO to various dsDNA oligonucleotides containing variations of the 5'-CTAG-3' binding motif by introducing inosine (I = inosine, 2-desaminoguanosine) and 5-methylcytosine ((me)C). A one- and two-dimensional NMR spectroscopy characterization yielded detailed structural information on the binding mode and for the well-defined TOTO-complexes competition experiments allowed determination of the relative binding strengths resulting from the various structural alterations. The experimentally observed base pair preference of TOTO in the palindromic sequences investigated is (me)CG > CG > CI > TA for the flanking base pair and (me)CI > CI > TA > CG > UA for the central base pair. The best binding site observed so far is the d(5-C(me)CIG-3')(2) site. This site is much more favorable than the d(5'-CTAG-3')(2) site formerly believed to be the best binding site. The present paper discusses these results in terms of different contributions to the binding affinity and offers some explanations for the site selectivity of TOTO.     

Structural comparison of anticancer drug-DNA complexes: adriamycin and daunomycin

The anticancer drugs adriamycin and daunomycin have each been crystallized with the DNA sequence d(CGATCG) and the three-dimensional structures of the complexes solved at 1.7- and 1.5-A resolution, respectively. These antitumor drugs have significantly different clinical properties, yet they differ chemically by only the additional hydroxyl at C14 of adriamycin. In these complexes the chromophore is intercalated at the CpG steps at either end of the DNA helix with the amino sugar extended into the minor groove. Solution of the structure of daunomycin bound to d(CGATCG) has made it possible to compare it with the previously reported structure of daunomycin bound to d(CGTACG). Although the two daunomycin complexes are similar, there is an interesting sequence dependence of the binding of the amino sugar to the A-T base pair outside the intercalation site. The complex of daunomycin with d(CGATCG) has tighter binding than the complex with d(CGTACG), leading us to infer a sequence preference in the binding of this anthracycline drug. The structures of daunomycin and adriamycin with d(CGATCG) are very similar. However, there are additional solvent interactions with the adriamycin C14 hydroxyl linking it to the DNA. Surprisingly, under the influence of the altered solvation, there is considerable difference in the conformation of spermine in these two complexes. The observed changes in the overall structures of the ternary complexes amplify the small chemical differences between these two antibiotics and provide a possible explanation for the significantly different clinical activities of these important drugs.     

Concerted intercalation and minor groove recognition of DNA by a homodimeric thiazole orange dye

The thiazole orange dye TOTO binds to double-stranded DNA (dsDNA) by a sequence selective bis-intercalation. Each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site, and the linker spans two base pairs in the minor groove. We have used one- and two-dimensional NMR spectroscopy to examine the dsDNA binding of an analogue of TOTO in which the linker has been modified to contain a bipyridyl group (viologen) that has minor groove binding properties. We have investigated the binding of this analogue, called TOTOBIPY, to three different dsDNA sequences containing a 5'-CTAG-3', a 5'-CTTAG-3', and a 5'-CTATAG-3' sites, respectively, demonstrating that TOTOBIPY prefers to span three base pairs. The many intermolecular NOE connectivities between TOTOBIPY and the d(CGCTTAGCG):d(CGCTAAGCG) oligonucleotide in the complex shows that the bipyridyl-containing linker is positioned in the minor groove and spans three base pairs. Consequently, we have succeeded in designing and synthesizing a ligand that recognizes an extended recognition sequence of dsDNA as the result of a concerted intercalation and minor groove binding mode.     

Modelling of the binding specificity in the interactions of cationic porphyrins with DNA.

A theoretical investigation is performed of the complexes of a tetracationic porphyrin, tetra-(4-N-methylpyridyl)-porphyrin, (T4MPyP), with the hexanucleotides d(CGCGCG)2 and d(TATATA)2, considering the possibility of both the intercalative and the groove binding interactions. These computations demonstrate that T4MPyP manifests a significant preference for intercalation in its complex with d(CGCGCG)2 but for non intercalative binding in the minor groove in its complex with d(TATATA)2. Such a dual binding behaviour of T4MPyP as a function of the sequence to which it is attached is fully consistent with available experimental data. It demonstrates that intercalation and groove binding may be viewed as two potential wells on a continuous energy surface. In agreement with experiment, the computations indicate that in the here considered case the deepest well is associated with intercalation.     

A theoretical investigation of the base sequence preferences of monointercalating polymethylene carboxamide derivatives 9-aminoacridine.

Theoretical computations are performed of the comparative binding affinities of five polymethylene carboxamide derivatives of 9-aminoacridine to a series of double-stranded hexanucleotides. The purpose of this investigation is to ascertain whether minor groove recognition of a guanine base adjacent to the intercalation site can occur, and be preferentially stabilized, for a given length of the polymethylene side chain, encompassing from n = 2 up to n = 6 methylene groups. For that purpose, several representative sequences were investigated, in which intercalation of the 9-aminoacridine chromophore occurred at a central d(CpG) or d(TpA) step. Investigated were the self-complementary sequences d(CGCGCG)2, d(GCCGGC)2, d(TATATA)2 and d(ATTAAT)2, as well as the 'mixed' sequences d(ACTAAT) .d(ATTAGT) and d(TGTATA). d(TATACA). For n = 3 up to n = 6, such a recognition was enabled only when the guanine base was located downstream of the intercalation site, i.e. with steps d(CGG) and d(TAG). It occurred by means of a bidentate interaction involving, on the one hand, H(N2) and N3 of the base, and, on the other hand, the carbonyl oxygen and the cis amino hydrogen of the terminal formamide moiety of the ligand. Because of the flexibility of the side chain, however, alternative binding modes were also found to occur competitively, involving backbone-only interactions of the side chain. On the basis of the present computations, upon binding to the sequence d(GCCGGC)2, an optimal value of n = 5 could be derived, with the corresponding acridine derivative eliciting both a significant prevalence of the bidentate over backbone only binding mode, and the most favourable energy balance within the investigated series. This privileged value of n = 5 is fully consistent with the experimental results of Markovits et al. and Gaugain et al. The very flexibility of the side chain, however, hampered any preferential recognition of a triplet sequence with a downstream guanine, such as d(CGG) or d(TAG), to be elicited over sequences such as d(TAA), d(TAT) or d(TAC).     

On the interaction of daunomycin with synthetic alternating DNAs: sequence specificity and polyelectrolyte effects on the intercalation equilibrium

The interaction of daunomycin with ctDNA and six purine–pyrimidine alternating poly-deoxynucleotides has been studied using fluorometric and uv-visible absorption methods. In the explored binding range of r > 0.05, the intercalation of the drug into the DNAs proved to be anticooperative, as indicated by the pronounced upward curvature of all the Scatchard plots obtained. The experimental data have been analyzed according to the recent theory of Friedman and Manning, which describes the polyelectrolyte effects on the site binding equilibria, drug intercalation included. We found that, accounting for the polyelectrolyte effects in the neighbor site exclusion model, the experimental data were nearly equally well described, in a wide range of binding ratios, by assuming the presence of sequence specificity effects (site size = 2 base pairs, exclusion parameter n = 1) or its absence (site size = 1 base pair, n = 1.7). The relevant results are as follows: (a) Daunomycin binds to all the DNAs considered with a stoichiometry of approximately 1 drug for every two base pairs. (b) The anticooperative nature of the interaction is essentially polyelectrolytic in origin. (c) The binding affinity shown by the drug for the different sites considered decreases in the order of Gm⁵C > AT > AC-GT > IC > GC > AU, indicating a stabilizing effect of the —CH₃ group in position 5 of the pyrimidines. (d) The extent of quenching of the intrinsic fluorescence of daunomycin in the presence of DNA is bound to the presence, at the intercalation site, of a guanine residue, since GC, Gm⁵C, and AC-GT sites induce a nearly total quenching, whereas AT, AU, and IC sites act only partially in this respect. The structural results obtained from the daunomycin-d[(CGTACG)]₂ crystal suggest that the 2-NH₂ group of guanine might be responsible for such a phenomenon. The influence of both the temperature and the ionic strength on the free energy of drug intercalation into ctDNA, poly[d(G-C)] : poly[d(G-C)], and poly[d(A-C)] : poly[d(G-T)] is examined and discussed.     

An NMR investigation of the binding of the anticancer drug actinomycin D to oligodeoxyribonucleotides with isolated 5'd(GC)3' binding sites

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with one GC binding site [d(ATATGCATAT) (1), d-(ATACGCGTAT) (2), and d(ATATACGCGTATAT) (3)] and with two GC sites [d(ATGCATGCAT) (4)]. At R = 1 (molar ratio of ActD to oligomer duplex) ActD caused a doubling of the number of imino proton signals at, and adjacent to, the GC binding site of 1. One of the G.C base pair signals shifted upfield while the other shifted downfield. Both of the signals for the A.T base pairs adjacent to the binding site shifted downfield. All imino proton signals of 2 and the longer sequence, 3, shifted upfield on binding of ActD to the GC site, indicating a sequence-dependent change in base stacking on complex formation. For both 1 and 2 addition of ActD resulted in a similar pattern of three downfield 31P NMR signals. The two most downfield signals have chemical shift and temperature dependence which are characteristic of phosphate groups at isolated intercalation sites. At R = 1 the ActD complex with 4 has very complex spectra with both upfield and downfield A.T and G.C imino signals. All these data were consistent with two 1:1 complexes with the unsymmetrical phenoxazone ring adopting both of the two possible orientations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethidium bromide interactions with DNA: an exploration of a classic DNA–ligand complex with unbiased molecular dynamics simulations

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)₂ also known as the Drew–Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 μs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.     

Site and sequence specificity of the daunomycin-DNA interaction

The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.     

A tentative model of the intercalative binding of the neocarzinostatin chromophore to double-stranded tetranucleotides.

Theoretical computations are performed of the intercalative binding of the neocarzinostatin chromophore (NCS) with the double-stranded oligonucleotides d(CGCG)2, d(GCGC)2, d(TATA)2 and d(ATAT)2. Minor groove binding is preferred over major groove binding. It is found that the long axis of the stacked naphtoate ring lies approximately parallel to the long axis of the base pairs of the intercalation site. The galactosamine ammonium group interacts with specific sites of the groove (O2/N3 of bases 2 and O1' of sugar S3), whereas the dodecadyine ring system wraps around the groove towards the backbone. An overall AT versus GC preference is derived. Intercalation in a central purine-(3', 5')-pyrimidine sequence appears to be preferred over that in a central pyrimidine-(3', 5')-purine sequence.     

The DNA sequence at echinomycin binding sites determines the structural changes induced by drug binding: NMR studies of echinomycin binding to [d(ACGTACGT)]2 and [d(TCGATCGA)]2

The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.     

Structural and Dynamical Studies of Humanin in Water and TFE/Water Mixture: A Molecular Dynamics Simulation

Summary

Proline-rich peptides are known to adopt preferentially the extended polyproline II (PPII) helical conformation, which is involved in several protein-protein recognition events. By resorting to molecular modelling techniques, we wished to investigate the extent to which PPII helices could be used for the formation of isohelical peptide-DNA complexes leading to the selective recognition of the major groove of B-DNA. For that purpose, we have grafted to a cationic intercalator, 9-amino-acridine, an oligopeptide having the sequence: Pro-Arg-Pro-Pro-Arg-Pro-Pro-Arg-Pro-Pro-Asp-Pro-Pro. Each residue in the sequence was set in the D configuration, to prevent enzymatic hydrolysis, and each Arg residue was designed to target O6/N7 of a guanine base following the intercalation site. The Asp residue was designed to target a cytosine base, whilst simultaneously forming a bidentate complex with the Arg three residues upstream. Energy-minimization, using the JUMNA procedure, led to the following conclusions: 1) major groove binding is favoured over minor groove or exclusive binding to the phosphates by large energy differences, of over 50 and 90 kcal/mole, respectively; 2) the two best bound sequences are those having three successive guanine bases on the same DNA strand, immediately adjacent to the intercalation site. Sequence d(CGGGC G), encountered in the Primer Binding Site of the HIV retrovirus, thus ranks amongst the best-bound sequences; 3) replacement of an individual guanine amongst the three ones upstream of the intercalation site, by an adenine base, weakens by > 6 kcal/mole the binding energetics; 4) the conformational rigidity of the DNA-bound PPII helix should enable for a modulation of the base sequence selectivity, by appropriate replacements of the Arg and Asp residues. Thus sequence CGGCAAG, also encountered in the HIV genome, could be targeted by an oligopeptide having the sequence Pro-Arg-Pro-Pro-Asp-Pro-Pro- Asn-Pro-Pro-Asn-Pro-Pro-Arg-Ala.     

NMR study of the drug-base overlap geometry in the dark complex of 8-methoxypsoralen and d(pApT)4

The dark binding of 8-methoxypsoralen (MOP) to d(pApT)4 was investigated by 270-MHz 1H nuclear magnetic resonance (NMR) spectra. The continuous high-field shifts of the MOP resonances by d(pApT)4 at low temperatures indicate fast exchange between free and bound drug. The limiting complexation shifts of the various MOP protons between 0.36 (CH3) and 1.20 ppm (H5) are in the range expected for an intercalation complex. The NMR line widths of the MOP ring protons vary with the square of the observed complexation shifts (maximum at H5), indicating a dominant effect of the fast exchange between free and bound drug. The corresponding kinetic parameters agree with the values previously reported for a variety of other intercalators. The observed exchange broadenings were also used as a criterion to limit the uncertainty connected with fast averaging of the signals of the drug in potential multiple binding modes: A qualitatively different pattern of broadenings (minimum at H5) is expected from fast exchange between the two binding modes related by the short 2-fold quasi-symmetry axis of MOP. The measured complexation shifts were compared to theoretical values calculated on the basis of coplanar intercalation with base pair arrangements derived from typical published intercalation site geometries. The standard deviation between observed and calculated shifts was considerably smaller for asymmetrical intercalation between the bases of the same strand (less than or equal to 0.11 ppm) than for symmetrical intercalation between the base pairs (greater than or equal to 0.28 ppm).(ABSTRACT TRUNCATED AT 250 WORDS)     

Base pair selectivity in the binding of copper (II) tetrakis (4-N-methylpyridyl)porphine to polynucleotides under closely packed conditions.

The base pair selectivity of the intercalative binding of the copper porphyrin, copper (II) tetrakis(4-N-methylpyridyl)porphine (Cu(II)TMpyP-4), to DNA has been investigated using a variety of DNA types and the synthetic polynucleotides poly(dG-dC)2 and poly(dA-dT)2. The studies utilize electron paramagnetic resonance of concentrated gels which are thought to mimic the closely packed state of nuclear DNA. The results indicate that intercalation of this porphyrin is preferred for sites containing two adjacent G-C base pairs, irrespective of sequence.