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.     

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.     

Solid-phase synthesis of acridine-based threading intercalator peptides

The preparation of a novel acridine-based amino acid is reported. This N-Alloc-protected monomer can be coupled and deprotected under solid-phase peptide synthesis procedures to create acridine peptide conjugates as potential threading intercalators. A peptide containing this novel amino acid undergoes spectral changes in the presence of duplex DNA and RNA consistent with intercalative binding.     

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.     

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.     

Half‐Intercalation Stabilizes Slipped Mispairing and Explains Genome Vulnerability to Frameshift Mutagenesis by Endogenous “Molecular Bookmarks”

Some 60 years ago chemicals that intercalate between base pairs of duplex DNA were found to amplify frameshift mutagenesis. Surprisingly, the robust induction of frameshifts by intercalators still lacks a mechanistic model, leaving this classic phenomenon annoyingly intractable. A promising idea of asymmetric half‐intercalation‐stabilizing frameshift intermediates during DNA synthesis has never been developed into a model. Instead, researchers of frameshift mutagenesis embraced the powerful slipped‐mispairing concept that unexpectedly struggled with the role of intercalators in frameshifting. It is proposed that the slipped mispairing and the half‐intercalation ideas are two sides of the same coin. Further, existing findings are reviewed to test predictions of the combined “half‐intercalation into the slipped‐mispairing intermediate” model against accumulated knowledge. The existence of potential endogenous intercalators and the phenomenon of “DNA bookmarks” reveal ample possibilities for natural frameshift mutagenisis in the cell. From this alarming perspective, it is discussed how the cell could prevent genome deterioration from frameshift mutagenesis.     

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.     

Absorption and fluorescence studies of the binding of the recA gene product from E. coli to single-stranded and double-stranded DNA. Ionic strength dependence

The binding of the recA gene product from E. coli to double-stranded and single-stranded nucleic acids has been investigated by following the change in melting temperature of duplex DNA and the fluorescence of single-stranded DNA or poly(dA) modified by reaction with chloroacetaldehyde. At low ionic strength, in the absence of Mg2+ ions, RecA protein binds preferentially to duplex DNA or poly(dA-dT). This leads to an increase of the DNA melting temperature. Stabilization of duplex DNA decreases when ionic strength or pH increases. In the presence of Mg2+ ions, preferential binding to single-stranded polynucleotides is observed. Precipitation occurs when duplex DNA begins to melt in the presence of RecA protein. From competition experiments, different single-stranded and double-stranded polydeoxynucleotides can be ranked according to their ability to bind RecA protein. Structural changes induced in nucleic acids upon RecA binding are discussed together with conformational changes induced in RecA protein upon magnesium binding.     

Cross-resistance of an amsacrine-resistant human leukemia line to topoisomerase II reactive DNA intercalating agents. Evidence for two topoisomerase II directed drug actions.

HL-60/AMSA is a human leukemia cell line that is 50-100-fold more resistant than its drug-sensitive HL-60 parent line to the cytotoxic actions of the DNA intercalator amsacrine (m-AMSA). HL-60/AMSA topoisomerase II is also resistant to the inhibitory actions of m-AMSA. HL-60/AMSA cells and topoisomerase II are cross-resistant to anthracycline and ellipticine intercalators but relatively sensitive to the nonintercalating topoisomerase II reactive epipodophyllotoxin etoposide. We now demonstrate that HL-60/AMSA and its topoisomerase II are cross-resistant to the DNA intercalators mitoxantrone and amonafide, thus strongly indicating that HL-60/AMSA and its topoisomerase II are resistant to topoisomerase II reactive intercalators but not to nonintercalators. At high concentrations, mitoxantrone and amonafide were also found to inhibit their own, m-AMSA's, and etoposide's abilities to stabilize topoisomerase II-DNA complexes. This appears to be due to the ability of these concentrations of mitoxantrone and amonafide to inhibit topoisomerase II mediated DNA strand passage at a point in the topoisomerization cycle prior to the acquisition of the enzyme-DNA configuration that yields DNA cleavage and topoisomerase II-DNA cross-links. In addition, amonafide can inhibit the cytotoxic actions of m-AMSA and etoposide. Taken together, these results suggest that the cytotoxicity of m-AMSA and etoposide is initiated primarily by the stabilization of the topoisomerase II-DNA complex. Other topoisomerase II reactive drugs may inhibit the enzyme at other steps in the topoisomerization cycle, particularly at elevated concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of intercalator binding on DNA triplex stability: correlation with effects on A-tract duplex structure

The triplex form of DNA is of interest because of a possible biological role as well as the potential therapeutic use of this structure. In this paper the stabilizing effects of two intercalating drugs, ethidium and the quinoxaline derivative 9-OH-B220, on DNA triplexes have been studied by thermal denaturation measurements. The corresponding duplex structures of the DNA triplex systems investigated are either A-tract or normal B-DNA. The largest increases in the triplex melting temperatures caused by the intercalators were found for sequences having A-tract duplex structures. Inserting a single base pair with an N2-amino group in the minor groove, e.g. a G-C pair, breaks up the A-tract duplex structure and also reduces the stabilizing effect of the drugs on the triplex melting temperatures. The large drug-induced increase in triplex melting temperature for complexes having an original duplex A-tract structure is correlated with a low initial melting point of the triplex, not with the triplex being unusually stable in the presence of the drug. Hence, we conclude that the large thermal stabilizing effect exhibited by ethidium and 9-OH-B220 on dTn.dAn-dTn triplexes is partly caused by the intercalators breaking up the intrinsic A-tract structure of the underlying duplex.     

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.     

Peptide nucleic acid-assisted topological labeling of duplex dna

Peptide nucleic acids (PNAs) are a family of synthetic polyamide mimics of nucleic acids that offer a variety of applications. Pyrimidine bis-PNAs can be used for rational design of novel interlocked DNA nanostructures, earring labels, representing locked pseudorotaxanes or locked catenanes. These structures are created through DNA ligase-mediated catenation of duplex DNA with a circularized oligonucleotide tag at a designated DNA site. The assembly is performed via formation of the PD-loop consisting of a pair of bis-PNA openers and the probe oligonucleotide. The openers locally expose one of the two strands of duplex DNA for hybridizing the probe, whose termini are complementary to the displaced DNA strand. After hybridization, they are in juxtaposition and can subsequently be linked by DNA ligase. As a result, a true topological link forms at a precise position on the DNA double helix yielding locked, earring-like label. DNA topological labeling can be done both in solution and, for longer templates, within the agarose gel plug. Accordingly, highly localized DNA detection with rolling circle amplification of hybridization signal and effective micromanipulations with DNA duplexes become possible through precise spatial positioning of various ligands on the DNA scaffold.     

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.     

Stabilization of DNA Triple Helices Using Conjugates of Oligonucleotides and Synthetic Ligands

The possibility is discussed of stabilizing a DNA triple helix by covalent conjugation to the third strand (through its terminal phosphate) of ligands that have affinity to double and triple helices. Two types of stabilizers are considered: minor groove binders based on oligopyrroles, and triplex-specific intercalators. As a target, a synthetic 29-mer duplex containing a natural polypurine sequence of the human immunodeficiency provirus was employed. The stabilization with minor groove binders requires several conditions to be respected: a sufficiently long linker capable of reaching the minor groove from the major groove, a specific double-stranded structure of the oligopyrrole fragment, and its in-phase fitness to the target sequence. The best stabilizers of a triplex were novel conjugates in which two parallel molecules containing six pyrrole units each are linked to the same 5"-phosphate of a 16-mer triplex-forming oligonucleotide. The stabilizing properties of these derivatives were comparable to those of benzoindoloquinoline (BIQ) intercalators attached to the terminal phosphate of triple-helix forming oligonucleotides.