Harnessing DNA intercalation

Numerous small molecules are known to bind to DNA through base pair intercalation. Fluorescent dyes commonly used for nucleic acid staining, such as ethidium, are familiar examples. Biological and physical studies of DNA intercalation have historically been motivated by mutation and drug discovery research. However, this same mode of binding is now being harnessed for the creation of novel molecular assemblies. Recent studies have used DNA scaffolds and intercalators to construct supramolecular assemblies that function as fluorescent 'nanotags' for cell labeling. Other studies have demonstrated how intercalators can be used to promote the formation of otherwise unstable nucleic acid assemblies. These applications illustrate how intercalators can be used to facilitate and expand DNA-based nanotechnology.     

Analysing DNA complexes by circular and linear dichroism

The application of linear and circular dichroism (LD and CD) in nucleic acid research id illustrated by recent results aimed at answering specific structural problem in the interaction of DNA with molecules of biological importance. We first consider the circumstances under which ligands, such as DAPI (4′, 6-diamidino-2-phenylindole), change their preferred binding mode in the minor groove to major groove binding or intercalation. As an extension of this problem we refer to the switch between groove binding and intercalation of structurally similar ligands such as ellipticines and trigonal ruthenium complexes. We also explore the use of LD and CD in the determination of the structure of the complex formed between the polynucleotide poly(dA) and the novel ‘peptide nucleic acid’, consisting of nucleic acid bases joined by a polyamide homomorphous with the deoxyribose-phosphate backbone of DNA. Finally, the structure and interaction of the recombination enzyme RecA with DNA is discussed, in particular the influence of the presence of the intercalators, groove binders or covalent DNA adducts.     

NMR structure determination of a modified DNA oligonucleotide containing a new intercalating nucleic acid

The intercalating nucleic acid (INA) presented in this paper is a novel 1-O-(1-pyrenylmethyl)glycerol DNA intercalator that induces high thermal affinity for complementary DNA. The duplex examined contained two INA intercalators, denoted X, inserted directly opposite each other: d(C(1)T(2)C(3)A(4)A(5)C(6)X(7)C(8)A(9)A(10)G(11)C(12)T(13)):d(A(14)G(15)C(16)T(17)-T(18)G(19)X(20)G(21)T(22)T(23)G(24)A(25)G(26)). Unlike most other nucleotide analogues, DNA with INA inserted has a lower affinity for hybridizing to complementary DNA with an INA inserted directly opposite than to complementary unmodified DNA. In this study we used two-dimensional (1)H NMR spectroscopy to determine a high-resolution solution structure of the weak INA-INA duplex. A modified ISPA approach was used to obtain interproton distance bounds from NOESY cross-peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Twenty final structures were generated for the duplex from a B-type DNA starting structure. The root-mean-square deviation (RMSD) of the coordinates for the 20 structures of the complex was 1.95 A. This rather large value, together with broad lines in the area of insertion, reflect the high degree of internal motion in the complex. The determination of the structure revealed that both intercalators were situated in the center of the helix, stacking with each other and the neighboring nucleobases. The intercalation of the INAs caused an unwinding of the helix in the insertion area, creating a ladderlike structure. The structural changes observed upon intercalation were mainly of local character; however, a broadening of the minor groove was found throughout the helix.     

Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity.

Although DNA helicases play important roles in the processing of DNA, little is known about the effects of DNA-interacting ligands on these helicases. Therefore, the effects of a wide variety of DNA-binding ligands on the unwinding and ATPase reactions catalyzed by Escherichia coli DNA helicase II were examined. DNA minor groove binders and simple DNA intercalators did not inhibit helicase II. However, DNA intercalators, such as mitoxantrone and nogalamycin, which position functionalities in the major groove upon binding duplex DNA, were potent inhibitors of helicase II. To determine the mechanism by which mitoxantrone inhibited helicase II, the unwinding and DNA-dependent ATPase activities of helicase II were measured using a spectrum of double- and single-stranded DNA substrates. Using either a 71-base pair (bp) M13mp7 partially duplexed DNA substrate or a 245-bp bluntended, fully duplexed DNA substrate, the apparent Ki value for inhibition by mitoxantrone of both the unwinding and ATPase reactions was approximately 1 microM for both substrates, suggesting that the mechanism of inhibition of helicase II by mitoxantrone is the same for both substrates and requires the presence of double-stranded structure. To strengthen this conclusion, the ability of mitoxantrone to inhibit the DNA-dependent ATPase activity of helicase II was determined using two single-stranded substrates, poly(dT) and the 245-bp substrate after heat denaturation. Using either substrate, mitoxantrone inhibited the ATPase activity of helicase II far less effectively. Thus, these results indicate that the intercalation of mitoxantrone into double-stranded DNA, with accompanying placement of functionalities in the major groove, generates a complex that impedes helicase II, resulting in both inhibition of ATP hydrolysis and unwinding activity. Furthermore, we report here that DNA-binding ligands inhibit the unwinding activity of helicases I and IV and Rep protein from E. coli, demonstrating that the inhibition observed for helicase II is not unique to this enzyme.     

Flexible docking of DNA fragments and actinocin derivatives

We have applied molecular docking methods to systems containing nucleic acids as targets and biologically active substances as ligands. The complexes of DNA fragments and actinocin derivatives with different lengths of aminoalkyl side chains were obtained by molecular docking. It was observed that actinocin derivatives could form energetically favourable complexes with DNA both as intercalators and minor groove binders. It was shown that small changes in the binding energy (～1?kcal/mol) could result in complexes with substantially different structure. The complexes of actinocin derivatives and DNA fragments were stabilized by hydrogen bonding upon intercalation and minor groove binding. It was found that the change of solvent-accessible surface area upon binding of the actinocin derivative to DNA linear increased with the growth of methylene groups' number in ligand side chains. The solvation energy change upon binding of actinocin derivatives to DNA calculated by the WSAS method was favourable in the case of small uncharged ligands and unfavourable for positively charged ligands.     

The Interaction Between the Novel Intercalator Diethidium Cation and B-Form DNA: a Theoretical Study

Abstract

This research is an effort to further understand the physicochemical interaction between the novel drug molecule diethidium (2,7-diamino 9-[2,7 diamino 10-nN- phenanthridium] 10- nN- phenanthridium) and its biological receptor DNA. The ultimate goal is the elucidation of this novel class of drugs as potential pharmaceutical agents. Understanding the physico- chemical properties of this drug as well as the mechanism by which it interacts with DNA should ultimately allow the rational design of novel anti-cancer or anti-viral drugs.

A novel binding structure for the diethidium cation to B-form DNA is herein described. Molecular modeling on the complex formed between diethidium and a dodecamer of double-stranded B-form DNA, CGCGAATTCGCG, has shown that this complex is indeed fully capable of participating in the formation of a stable intercalation site. It was expected that diethidium would have a mechanism of intercalation significantly different from other classical intercalators because a) Its structure, that of two perpendicular planes, each known to have excellent intercalation properties, is novel b) The linker region length is zero c) The tilt between the two planes of the drug matches the geometry of the space available to this drug in the major groove.

We have studied the complex formed when diethidium enters the central site of the B-DNA dodecamer through the major groove. The complex forms several classes of intercalation structures, which are all stable and vary from “partially intercalate” to “fully intercalated”. Multiple minimizations show the drug to be very mobile within the intercalation site. Further, some structures show organization and concomitant stiffening of the DNA above the intercalation site, with a disorganization and disruption of the regular B-DNA structure immediately below the intercalation site. This particular phenomena may be expected to lead to significantly different physicochemical properties for the diethidium complex with respect to other known intercalators, because this sort of vectorial difference in structure above and below the site of intercalation is unknown in existing intercalators, as far as the authors are aware. In addition, we expect the mechanism of interaction between drug and DNA to be described by “direct ligand transfer”, wherein the drug is transferred from duplex DNA to duplex DNA without re-entering the solvent.¹

This work is the first instance known to the authors of a novel drug entity that was deduced solely by mathematical reasoning ² and described subsequently by computational methods. Evidence that diethidium should interact with its target site DNA differently from other known intercalators is strong.     

Virtual screening,synthesis and biological evaluation of DNA intercalating antiviral agents

This paper describes computer-aided design of new anti-viral agents against Vaccinia virus (VACV) potentially acting as nucleic acid intercalators. Earlier obtained experimental data for DNA intercalation affinities and activities against Vesicular stomatitis virus (VSV) have been used to build, respectively, pharmacophore and QSAR models. These models were used for virtual screening of a database of 245 molecules generated around typical scaffolds of known DNA intercalators. This resulted in 12 hits which then were synthesized and tested for antiviral activity against VaV together with 43 compounds earlier studied against VSV. Two compounds displaying high antiviral activity against VaV and low cytotoxicity were selected for further antiviral activity investigations.     

Unwinding of unnatural substrates by a DNA helicase

Helicases separate double-stranded DNA into single-stranded DNA intermediates that are required during replication and recombination. These enzymes are believed to transduce free energy available from ATPase activity to unwind the duplex and translocate along the nucleic acid lattice. The nature of enzyme-substrate interactions between helicases and duplex DNA substrates has not been well-defined. Most helicases require a single-stranded DNA overhang adjacent to duplex DNA in order to initiate unwinding. The strand containing the overhang is referred to as the loading strand whereas the complementary strand is referred to as the displaced strand. We have investigated the interactions between a DNA helicase and the DNA substrate by replacing the displaced strand with a nucleic acid mimic, peptide nucleic acid (PNA). PNA is capable of forming duplex structures with DNA according to Watson-Crick base pairing rules, but contains a N-(2-aminoethyl)glycine backbone in place of the deoxyribose phosphates. The PNA-DNA hybrids had higher melting temperatures than their DNA-DNA counterparts. Dda helicase, from bacteriophage T4, was able to unwind the DNA-PNA substrates at similar rates as DNA-DNA substrates. The results indicate that the rate-limiting step for unwinding is relatively insensitive to the chemical nature of the displaced strand and the thermal stability of oligonucleotide substrates.     

Interactions of molecules with nucleic acids. XI. Generalization of techniques to generate nucleic acid structures with applications to intercalation sites and kinked structures

A generalized procedure to generate nucleic acid structures is presented. In this procedure, the bases of a base pair are oriented first for characterization of particular DNA receptor sites. The resultant sites are then used in the study of specific molecule–DNA interactions. For example, intercalation sites, kinked DNA, and twisted and tilted bases are envisioned. Alterations of structures via anti → syn orientations of bases, as well as crankshaft motion about collinear bonds, provide additional conformations without disrupting the overall backbone structure. These approaches to the generation of nucleic acid structures are envisioned as required in studies of the intercalation phenomenon, minor adjustments of DNA to accommodate denaturation, binding of carcinogens to DNA, complex formation of transition metals with DNA, and antitumor agents as ligands. For these base-pair and base orientations, backbone orientations are calculated by the AGNAS technique to yield physically meaningful conformations, namely, those conformations for which nonbonded contacts are favourable. A procedure is presented to generate dimer duplex units that are physically meaningful and to assemble these units into a polynucleotide duplex. Double helices that begin with B-DNA, undergo a transition to one of the above-mentioned receptor sites, and return to B-DNA can be assembled from a catalog of dimer duplexes. Stereographic projections of the various receptor sites already being used to model binding to DNA are presented.     

A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids.     

Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents

Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.     

1H-nuclear magnetic resonance of intercalators and rGCA: a potential mutagenicity probe

Variable temperature 1H-nuclear magnetic resonance (NMR) has been used to study the interaction of the RNA trimer, GpCpA, with the intercalators ethidium bromide and the acridine derivatives; proflavin, 9-amino-acridine, acridine orange, acridine yellow and acriflavin. The complexes formed were studied at nucleic acid to drug ratios of 1:1 and 5:1, the latter being useful in defining the effects of structural variation in the acridine series and in determining the site of intercalation. All the intercalators greatly stabilized the oligonucleotide duplex, the average melting temperature (Tm) increasing by up to 30 degrees C. Significant changes in individual Tms and chemical shifts were observed for all the GpCpA protons. 9-Amino-acridine and acriflavin did not stabilize the GpCpA duplex as substantially as the other acridine derivatives. It is suggested that this intercalator:GpCpA system, and its associated NMR-derived Tm, is a useful physical probe for potential mutagens.     

Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization

A new method for helicase-catalyzed DNA unwinding is described. This assay takes advantage of the substantial change in fluorescence polarization (FP) upon helicase binding and DNA unwinding. The low anisotropy value, due to the fast tumbling of the free oligonucleotide in solution, increases abruptly upon binding of helicase to the fluorescein-labeled oligonucleotide. The high anisotropy of the helicase– DNA complex decreases as the fluorescein-labeled oligonucleotide is released from the complex through helicase-catalyzed DNA unwinding. This FP signal can be measured in real time by fluorescent spectroscopy. This assay can simultaneously monitor DNA binding and helicase-catalyzed DNA unwinding. It can also be used to determine the polarity in DNA unwinding mediated by helicase. This FP assay should facilitate the study of the mechanism by which helicase unwinds duplex DNA, and also aid in screening for helicase inhibitors, which are of growing interest as potential anticancer agents.     

Interaction of unfused tricyclic aromatic cations with DNA: a new class of intercalators

Unfused tricyclic aromatic ring systems 1-6 with one or two cationic side chains have been synthesized and their interactions with DNA and synthetic polymers probed with a variety of techniques. Molecular mechanics calculations indicate that the torsional angle between ring planes in the minimum energy conformation of the tricyclic molecules can range from 0 degree to as high as 50 degrees depending on the type of rings and substituents. Viscometric titrations with linear and supercoiled DNA, linear dichroism, and NMR studies indicated that all compounds with torsional angles of approximately 20 degrees or less bind to DNA by intercalation. The more highly twisted intercalators caused significant perturbation of DNA structure. Unfused intercalators with twist angles of approximately 20 degrees have reduced binding constants, suggesting that they could not form an optimum interaction with the DNA base pairs. Unfused intercalators with twist less than 20 degrees formed strong complexes with DNA. The structures of these unfused intercalators are more analogous to typical groove-binding molecules, and an analysis of their interaction with DNA provides a better understanding of the subtle differences between intercalation and groove-binding modes for aromatic cations. The results indicate that intercalation and groove-binding modes should be viewed as two potential wells on a continuous energy surface. The results also suggest design strategies for intercalators that can optimally complement DNA base pair propeller twist or that can induce bends in DNA at the intercalation site.     

Analysis of the DNA substrate specificity of the human BACH1 helicase associated with breast cancer

We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ' ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ' ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination.     

Interactions of cyclic and non-cyclic naphthalene diimide derivatives with different nucleic acids

Recently, strategy based on stabilization of G-quadruplex telomeric DNA by small organic molecule has been realized by naphthalene diimide derivatives (NDIs). At the same time NDIs bind to DNA duplex as threading intercalators. Here we present cyclic derivative of naphthalene diimide (ligand 1) as DNA-binding ligand with ability to recognition of different structures of telomeric G-quadruplexes and ability to bis-intercalate to double-stranded helixes. The results have been compared to non-cyclic derivative (ligand 2) and revealed that preferential binding of ligands to nucleic acids strongly depends on their topology and structural features of ligands.