Anticooperative Binding Governs the Mechanics of Ethidium-Complexed DNA

DNA intercalators bind nucleic acids by stacking between adjacent basepairs. This causes a considerable elongation of the DNA backbone as well as untwisting of the double helix. In the past few years, single-molecule mechanical experiments have become a common tool to characterize these deformations and to quantify important parameters of the intercalation process. Parameter extraction typically relies on the neighbor-exclusion model, in which a bound intercalator prevents intercalation into adjacent sites. Here, we challenge the neighbor-exclusion model by carefully quantifying and modeling the force-extension and twisting behavior of single ethidium-complexed DNA molecules. We show that only an anticooperative ethidium binding that allows for a disfavored but nonetheless possible intercalation into nearest-neighbor sites can consistently describe the mechanical behavior of intercalator-bound DNA. At high ethidium concentrations and elevated mechanical stress, this causes an almost complete occupation of nearest-neighbor sites and almost a doubling of the DNA contour length. We furthermore show that intercalation into nearest-neighbor sites needs to be considered when estimating intercalator parameters from zero-stress elongation and twisting data. We think that the proposed anticooperative binding mechanism may also be applicable to other intercalating molecules.     

Increasing the analytical sensitivity by oligonucleotides modified with para- and ortho-twisted intercalating nucleic acids--TINA

The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5' and 3' termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch--in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems.     

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.     

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.     

Design and synthesis of threading intercalators to target DNA

Threading intercalators are high affinity DNA binding agents that bind by inserting a chromophore into the duplex and locating one group in each groove. The first threading intercalators that can be conjugated to acids, sulfonic acids and peptides to target them to duplex DNA are described, based upon the well studied acridine-3- or 4-carboxamides. Cellular uptake of the parent acridine is rapid and it can be visualized in the nucleus of cells. Both the parent compounds and their conjugates maintain antitumor activity.     

T-T base mismatches enhance drug binding at the branch site in a four-arm DNA junction.

Base mismatches--non Watson-Crick pairing between bases--can arise in duplex DNA as a consequence of mutational events or by recombination. In a duplex, the sequence of the two bases involved, and those flanking the site of mismatch, determines the local structure and extent of destabilization of the helix. Base mismatches can arise also in recombination of nonhomologous strands, and their occurrence in Holliday recombination intermediates can influence the outcome of general or specialized recombination events. We have previously reported that the branch site in a DNA junction can interact selectively with a variety of ligands. Here we describe the thermodynamics of junctions containing T-T mismatches flanking the branch and show that these structures bind methidium and other intercalators with higher affinity than junctions lacking mismatches.     

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.     

Design of molecules that specifically recognize and cleave apurinic sites in DNA

We have prepared a series of a tailor-made molecules that recognize and cleave DNA at apurinic sites in vitro. These molecules incorporate in their structure different units designed for specific function: an intercalator for DNA binding, an nucleic base for abasic site recognition and a linking chain of variable length and nature (including amino and/or amido functions). The cleavage efficiency of the molecules can be modulated by varying successively the nature of the intercalating agent, the nucleic base and the chain. All molecules bind to native calf thymus DNA with binding constants ranging from 10⁴ to 10⁶ M^?1. Their cleavage activity was determined on plasmid DNA (pBR 322) containing 1.8 AP-sites per DNA-molecule. The minimum requirements for cleavage are the presence of the three units, the intercalator, the nucleic base and at least one amino function in the chain. The most efficient molecules cleaved plasmid DNA at nanomolar concentrations. Enzymatic experiments on the termini generated after cleavage of AP-DNA suggest a strand break induced by a β-elimination reaction. In order to get insight into the mode of action (efficiency, selectivity, interaction), we have used synthetic oligonucleotides containing either a true abasic site at a determined position to analyse the cleavage parameters of the synthetic molecules by HPLC or a chemically stable along (tetrahydrofuran) of the abasic site for high field ¹H NMR spectrometry and footprinting experiments. All results are consistent with a β-elimination mechanism in which each constituent of the molecule exerts a specific function as indicated in the scheme: DNA targeting, abasic site nucleases and can be used advantageously as substitutes for the natural enzyme for in vitro cleavage of AP-sites containing DNA.     

Linkage structures strongly influence the binding cooperativity of DNA intercalators conjugated to triplex forming oligonucleotides.

Conjugation of DNA intercalators to triple helix forming oligodeoxynucleotides (ODN's) can enhance ODN binding properties and consequently their potential ability to modulate gene expression. To test the hypothesis that linkage structure could strongly influence the binding enhancement of intercalator conjugation with triplex forming ODN's, we have used a model system to investigate binding avidity of short oligomers conjugated to DNA intercalators through various linkages. Using a dA10.T10 target sequence imbedded in a 20 bp duplex, binding avidities of a T10 ODN joined to the DNA intercalator 6,9-diamino, 3-methoxy acridine (DAMA) by 8 different 5' linkages were measured using an electrophoretic mobility shift assay. Although unmodified T10 has a very limited capacity for stable binding under these conditions (apparent Kd > 250 microM at 4 degrees C), conjugation to DAMA using flexible linkers of certain lengths and chemical compositions greatly enhanced binding (Kd of 1 microM at 4 degrees C). Other linkers, however, modestly enhanced binding or had no effect on binding at all. Thus, the length, flexibility, and chemical composition of linker structures all substantially influence intercalator conjugated oligodeoxynucleotide binding avidity.     

Sensitive and label-free biosensing of RNA with predicted secondary structures by a triplex affinity capture method

A novel biosensing approach for the label-free detection of nucleic acid sequences of short and large lengths has been implemented, with special emphasis on targeting RNA sequences with secondary structures. The approach is based on selecting 8-aminoadenine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. With this strategy, we have detected short DNA sequences (32-mer) and purified RNA (103-mer) at the femtomol level in a few minutes in an easy and level-free way. This approach is particularly suitable for the detection of RNA molecules with predicted secondary structures, reaching a limit of detection of 50 fmol without any label or amplification steps. Our methodology has shown a marked enhancement for the detection (18% for short DNA and 54% for RNA), when compared with the conventional duplex approach, highlighting the large difficulty of the duplex approach to detect nucleic acid sequences, especially those exhibiting stable secondary structures. We believe that our strategy could be of great interest to the RNA field.     

On the fidelity of DNA replication. Mechanisms of misincorporation by intercalating agents

Two distinct mechanisms of action for intercalating agents have been delineated: one leading to the production of frameshift misincorporations and the other leading to the production of single-base substitutions. Addition misincorporations are competitive with respect to DNA template (a measure of classical intercalation) but are not competitive with respect to deoxynucleotide substrates. Single-base substitutions are not competitive with template, polymerase, or deoxynucleotide as tested individually, but are proportional to the absolute drug concentration, indicating a ternary complex involving intercalator, polymerase, and template. Increased frequencies of single-base substitutions have not been considered as a general property of intercalators. Using a mutant phi X174 DNA, we demonstrate that intercalators also induce single-base substitutions with natural DNA templates. Reversion of am3 phi X174 DNA occurs only by single-base substitutions at position 587; this is increased 8-fold when the DNA is copied in vitro in the presence of intercalators.     

RNA-ligand interactions: affinity and specificity of aminoglycoside dimers and acridine conjugates to the HIV-1 Rev response element

Semisynthetic aminoglycoside derivatives may provide a means to selectively target viral RNA sites, including the HIV-1 Rev response element (RRE). The design, synthesis, and evaluation of derivatives based upon neomycin B, kanamycin A, and tobramycin conjugates of 9-aminoacridine are presented. To evaluate the importance of the acridine moiety, a series of dimeric aminoglycosides as well as unmodified "monomeric" aminoglycosides have also been evaluated for their nucleic acid affinity and specificity. Fluorescence-based binding assays that use ethidium bromide or Rev peptide displacement are used to quantify the affinities of these compounds to various nucleic acids, including the RRE, tRNA, and duplex DNA. All the modified aminoglycosides exhibit a high affinity for the Rev binding site on the RRE (K(d) 相似文献   

Design and analysis of RNA structure-specific agents as potential antivirals

A number of pathogenic RNA viruses, such as HIV-1, have extensive folded RNA conformations with imperfect A-form duplexes that are essential for virus function, and could serve as targets for structure-specific antiviral drugs. A method for the discovery of such drugs involves evaluation of the interactions with RNA of a wide variety of compounds that are known to bind to nucleic acids by different mechanisms. This approach has been initiated by using corresponding sequence RNA and DNA polymers as initial test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA do not have significant interactions with RNA. Polycations, however, can show significant RNA affinity and binding selectivity, probably through complex formation in the RNA major groove. Some intercalators and a group of diphenylfuran cations have strong interactions with RNA that are very dependent on compound structure. RNA hairpin model systems for the RRE binding site of HIV-1 Rev protein were constructed for more detailed investigations. The diphenylfuran cations bind strongly to RRE and selectively inhibit Rev binding. CD, NMR, and fluorescence binding studies indicate that the active compounds bind in the internal loop region of RRE (with binding constants >10⁷M⁻¹), and cause a conformational change in the RNA. None of the standard nucleic acid binding modes appears to fit the results for complexes of the active compounds with RRE, and it is proposed that the diphenylfuran system threads through the internal loop region of RRE. Such a model allows contacts of the furan cationic substituents with both grooves of RRE in addition to the intercalation interactions with the bases.     

Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase.

An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-microns DEAE anion-exchange resin capable of chromatographing nucleic acids greater than 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 M potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When [14C]doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 M sodium chloride at 50 degrees C for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single-stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA (34 pmol/100 micrograms), and the highest levels were recorded with oligonucleotides (243 pmol/100 micrograms). The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 microM [14C]doxorubicin (18.6 pmol/100 micrograms). Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques.     

Selective binding of tumor suppressor p53 protein to topologically constrained DNA: Modulation by intercalative drugs

Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.