Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation

Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.     

Specific versus nonspecific binding of cationic PNAs to duplex DNA

Although peptide nucleic acids (PNAs) are neutral by themselves, they are usually appended with positively charged lysine residues to increase their solubility and binding affinity for nucleic acid targets. Thus obtained cationic PNAs very effectively interact with the designated duplex DNA targets in a sequence-specific manner forming strand-invasion complexes. We report on the study of the nonspecific effects in the kinetics of formation of sequence-specific PNA-DNA complexes. We find that in a typical range of salt concentrations used when working with strand-invading PNAs (10-20 mM NaCl) the PNA binding rates essentially do not depend on the presence of nontarget DNA in the reaction mixture. However, at lower salt concentrations (<10 mM NaCl), the rates of PNA binding to DNA targets are significantly slowed down by the excess of unrelated DNA. This effect of nontarget DNA arises from depleting the concentration of free PNA capable of interacting with DNA target due to adhesion of positively charged PNA molecules on the negatively charged DNA duplex. As expected, the nonspecific electrostatic effects are more pronounced for more charged PNAs. We propose a simple model quantitatively describing all major features of the observed phenomenon. This understanding is important for design of and manipulation with the DNA-binding polycationic ligands in general and PNA-based drugs in particular.     

Towards echinomycin mimetics by grafting quinoxaline residues on glycophane scaffolds

Echinomycin is a natural depsipeptide, which is a bisintercalator, inserting quinoxaline units preferentially adjacent to CG base pairs of DNA. Herein the design and synthesis of echinomycin mimetics based on grafting of two quinoxaline residues onto a macrocyclic scaffold (glycophane) is addressed. Binding of the compounds to calf-thymus DNA was studied using UV-vis and steady state fluorescence spectroscopy, as well as thermal denaturation. An interesting observation was enhancement of fluorescence emission for the peptidomimetics on binding to DNA, which contrasted with observations for echinomycin. Molecular dynamics simulations were exploited to explore in more detail if bis-intercalation to DNA was possible for one of the glycophanes. Bis-intercalating echinomycin complexes with DNA were found to be stable during 20ns simulations at 298K. However, the MD simulations of a glycophane complexed with a DNA octamer displayed very different behaviour to echinomycin and its quinoxaline units were found to rapidly migrate out from the intercalation site. Release of bis-intercalation strain occurred with only one of the quinoxaline chromophores remaining intercalated throughout the simulation. The distance between the quinoxaline residues in the glycophane at the end of the MD simulation was 7.3-7.5?, whereas in echinomycin, the distance between the residues was ～11?, suggesting that longer glycophane scaffolds would be required to generate bis-intercalating echinomycin mimetics.     

Interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments containing isolated ligand binding sites

We have examined the interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments which contain isolated ligand binding sites. A 145 base pair fragment was prepared on the basis of the sequence of tyrT DNA, which contained no CpG or (A/T)(4) binding sites for these ligands. Isolated binding sites were introduced into this fragment at discrete locations where the minor groove is known to face toward or away from the protein core when reconstituted onto nucleosome core particles. The interaction of ligands with target sites on these nucleosomal DNA fragments was assessed by DNase I footprinting. We find that Hoechst 33258 can bind to single nucleosomal sites which face both toward and away from the protein core, without affecting the nucleosome structure. Hoechst binding is also observed on nucleosomal fragments which contain two or more drug binding sites, though in these cases the footprints are accompanied by the presence of new cleavage products in positions which suggest that the ligand has caused a proportion of the DNA molecules to adopt a new rotational positioning on the protein surface. Hoechst 33258 does not affect nucleosome reconstitution with any of these fragments. In contrast, the bifunctional intercalating antibiotic echinomycin is not able to bind to single nucleosomal CpG sites. Echinomycin footprints are observed on nucleosomal fragments containing two or more CpG sites, but there are no changes in the cleavage patterns in the remainder of the fragment. Echinomycin abolishes nucleosome reconstitution when included in the reconstitution mixture.     

The 2-amino group of guanine is absolutely required for specific binding of the anti-cancer antibiotic echinomycin to DNA.

The 2-amino group of guanine is believed to be a critical determinant of potential DNA binding sites for echinomycin and related quinoxaline antibiotics. In order to probe its importance directly we have studied the interaction between echinomycin and DNA species in which guanine N(2) is deleted by virtue of substitution of inosine for guanosine residues. The polymerase chain reaction was used to prepare inosine-substituted DNA. Binding of echinomycin, assessed by DNAse I footprinting, was practically abolished by incorporation of inosine into one or both strands of DNA. We conclude that both the purines in the preferred CpG binding site need to bear a 2-amino group to interact with echinomycin.     

Transferring the purine 2-amino group from guanines to adenines in DNA changes the sequence-specific binding of antibiotics.

The proposition that the 2-amino group of guanine plays a critical role in determining how antibiotics recognise their binding sites in DNA has been tested by relocating it, using tyrT DNA derivative molecules substituted with inosine plus 2,6-diaminopurine (DAP). Irrespective of their mode of interaction with DNA, such GC-specific antibiotics as actinomycin, echinomycin, mithramycin and chromomycin find new binding sites associated with DAP-containing sequences and are excluded from former canonical sites containing I.C base pairs. The converse is found to be the case for a group of normally AT-selective ligands which bind in the minor groove of the helix, such as netropsin: their preferred sites become shifted to IC-rich clusters. Thus the binding sites of all these antibiotics strictly follow the placement of the purine 2-amino group, which accordingly must serve as both a positive and negative effector. The footprinting profile of the 'threading' intercalator nogalamycin is potentiated in DAP plus inosine-substituted DNA but otherwise remains much the same as seen with natural DNA. The interaction of echinomycin with sites containing the TpDAP step in doubly substituted DNA appears much stronger than its interaction with CpG-containing sites in natural DNA.     

The effects of local DNA sequence on the interaction of ligands with their preferred binding sites

We have examined the effects of local DNA sequence on the interaction of distamycin, Hoechst 33258, echinomycin, actinomycin and mithramycin with their preferred binding sites using a series of DNA fragments that contain every symmetrical hexanucleotide sequence. In several instances we find that the affinity for the ligands' preferred binding sites is affected by the hexanucleotide context in which they are located. The AT-selective minor groove binding ligand Hoechst 33258 shows a 200-fold difference in binding to the 16 different X(A/T)(4)Y sites; the strongest binding is to AAATTT and the weakest is to (G/C)TTAA(C/G). Although TTAA is generally a poor binding site, ATTAAT is better than TTTAAA and they are both much better than GTTAAC and CTTAAG. Similarly, TTATAA and ATATAT are better binding sites than GTATAC and CTATAG. In contrast, distamycin shows less discrimination between the various X(A/T)(4)Y sites, with a 20-fold difference between the best [(A/T)AATT(T/A)] and worst [GATATC and (G/C)TTAA(C/G)] sites. Although actinomycin binds to GpC it shows little or no interaction with any of the GGCC sites, yet shows only a six-fold variation in affinities for the other XYGCXY sites. Echinomycin binds to CpG yet shows no binding to TTCGAA, TGCGCA and AGCGCT, while the best binding is to AACGTT. The tetranucleotides CCGG and ACGT produce consistently good binding sites, irrespective of the surrounding sequences, while the interaction with TCGA and GCGC is sensitive to the hexanucleotide context. Hexanucleotides with a central GCGC, flanked by A and T are weaker echinomycin sites than those flanked by G and C, especially CGCGCG. The best X(G/C)(4)Y binding sites for mithramycin were located at AGCGCT and GGGCCC, and the worst at CCCGGG and TCCGGA. These footprinting fragments are valuable tools for comparing the binding of ligands to all the potential symmetrical hexanucleotides and provide insights into the effects of local DNA sequence on ligand-DNA interactions.     

Interaction between synthetic analogues of quinoxaline antibiotics and nucleic acids. Changes in mechanism and specificity related to structural alterations.

The interaction with DNA of six chemically synthesized derivatives of the quinoxaline antibiotics was investigated. Five of the compounds bound only weakly to DNA or not at all; for these substances spectrophotometric measurements, sedimentation studies with closed circular duplex bacteriophage-PM2 DNA and thermal-denaturation profiles were used to determine limits fot the binding constants. No interaction could be detected with two products of degradation of echinomycin (quinomycin A), one of which, echinomycinic acid dimethyl ester, had the lactone linkages opened, whereas the other retained an intact octapeptide ring but had a broken cross-bridge. The other compounds studied were des-N-tetramethyl-triostin A ('TANDEM') and its derivatives. A derivative of 'TANDEM' IN WHICH benzyloxycarbonyl moieties replace both quinoxaline chromophores had binding constants to nucelic acids in the range 10(2)--10(3)-1, whereas no interaction could be detected for a benzyloxycarbonyl derivative that, in addition, had the cross-bridge broken. The derivative of 'TANDEM' with L-serine in place of D-serine in both positions showed no detectable interaction with Clostridium perfringens DNA, whereas the binding constant to poly(dA-dT) was approx 2 X 10(3)M-1. 'TANDEM' itself bound strongly to DNA, and the bathochromic and hypochromic shifts in its u.v.-absorption spectrum in the presence of DNA were similar to those seen with echinomycin. From the effect on the sedimentation coefficient of closed circular duplex bacteriophage-PM2 DNA the mechanism of binding was shown to involve bifunctional intercalation, typical of the naturally occurring quinoxaline antibiotics. Solvent-partition analysis was used to determine binding constants for the interaction between 'TANDEM' and a variety of natural and synthetic DNA species. The pattern of specificity thus revealed differed markedly from that previously found with the naturally occurring quinoxaline antibiotics. Most striking was the evident large preference for (A + T)-rich DNA species, in complete contrast with echinomycin and triostin A. The highest binding constant was found for poly(dA-dT), the interaction with which appeared highly co-operative in character. The conformations adopted by those quinoxaline compounds that bind strongly to DNA were examined withe aid of molecular models on the basis of results derived from n.m.r. and computer studies. It appears that the observed patterns of base-sequence specificity are determined, at least in part, by the structure and conformation of the sulphur-containing cross-bridge.     

Development of a self-assembling nuclear targeting vector system based on the tetracycline repressor protein

The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR.tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4- and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2-8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetRNLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.     

The binding of echinomycin to deoxyribonucleic acid.

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.     

Combined triplex/duplex invasion of double-stranded DNA by "tail-clamp" peptide nucleic acid

"Tail-clamp" PNAs composed of a short (hexamer) homopyrimidine triplex forming domain and a (decamer) mixed sequence duplex forming extension have been designed. Tail-clamp PNAs display significantly increased binding to single-stranded DNA compared with PNAs lacking a duplex-forming extension as determined by T(m) measurements. Binding to double-stranded (ds) DNA occurred by combined triplex and duplex invasion as analyzed by permanganate probing. Furthermore, C(50) measurements revealed that tail-clamp PNAs consistently bound the dsDNA target more efficiently, and kinetics experiments revealed that this was due to a dramatically reduced dissociation rate of such complexes. Increasing the PNA net charge also increased binding efficiency, but unexpectedly, this increase was much more pronounced for tailless-clamp PNAs than for tail-clamp PNAs. Finally, shortening the tail-clamp PNA triplex invasion moiety to five residues was feasible, but four bases were not sufficient to yield detectable dsDNA binding. The results validate the tail-clamp PNA concept and expand the applications of the P-loop technology.     

Optimization of label-free DNA detection with electrochemical impedance spectroscopy using PNA probes

DNA biosensors, especially those based upon detection of the intrinsic negative charge of target DNA, can be greatly improved by the use of uncharged peptide nucleic acid (PNA) probes. Hybridization causes an increased electrostatic barrier for the negatively charged ferri/ferrocyanide redox couple, resulting in an increase in charge transfer resistance R(ct) that is measured using electrochemical impedance spectroscopy. We report on the optimization of PNA probe surface density by the simultaneous co-immobilization of thiol-modified probes and mercaptohexanol, with the PNA surface density controlled by the thiol mole ratio in solution. Maximum R(ct) change upon hybridization is obtained with 10% PNA mole fraction. The effect of the measurement buffer ionic strength is investigated. The electrostatic barrier for charge transfer to the ferri/ferrocyanide redox couple is approximately independent of ionic strength with PNA probes, but greatly increases with decreasing ionic strength, after hybridization with target DNA. This significantly enhances the R(ct) change upon hybridization. The optimization of PNA surface density and measurement buffer ionic strength leads to a 385-fold increase in R(ct) upon hybridization, a factor of 100 larger than previously reported results using either PNA or DNA probes.     

Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.     

Binding of quinoline analogues of echinomycin to deoxyribonucleic acid. Role of the chromophores.

Two novel antibiotics were isolated, designated compounds 1QN and 2QN respectively, having quinoline rings in place of one or both of the quinoxaline chromophores of echinomycin. Each removes and reverses the supercoiling of closed circular duplex DNA from bacteriophage PM2 in the fashion characteristic of intercalating drugs. For compound 1QN, the unwinding angle at I0.01 is almost twice that of ethidium, whereas for compound 2QN the value is indistinguishable from that of ethidium. Binding of both analogues produced changes in the viscosity of sonicated rod-like DNA fragments corresponding to double the helix extension found with ethidium, a feature characteristic of bifunctional intercalation by quinoxaline antibiotics. These results suggest that both compounds 1QN and 2QN behave as bifunctional intercalators but that compound 2QN produces only half the helix unwinding seen with compound 1QN and the natural quinoxalines. Binding curves for the interaction of both analogues with a variety of synthetic and naturally occurring nucleic acids were determined by solvent-partition analysis. Values for compound 2QN were also obtained by a fluorimetric method and found to agree well with the solvent-partition measurements. Compound 1QN bound most tightly to Micrococcus lysodeikticus DNA and, like echinomycin, exhibited a broad preference for (G + C)-rich DNA species. For compound 2QN no marked (G + C) preference was indicated, and the tightest binding among the natural DNA species studied was found with DNA from Escherichia coli. The two analogues also displayed different patterns of specificity in their interaction with synthetic nucleic acids. Compound 2QN bound to poly(dA-dT) slightly more tightly than to poly-(dG-dC), whereas compound 1QN displayed a large (approx. 11-fold) preference in the opposite sense. There was evidence of co-operativity in the binding to poly(dA-dT). It may be concluded that the chromophore moieties play an active role in determining the capacity of quinomycin antibiotics to recognize and bind selectively to specific sequences in DNA.     

Study of the toxicity and antiblastoma activity of antibiotics echinomycin and 6270 in complexes with DNA

Studies with the use of intact inbred albino mice showed that in intravenous administration the acute toxicity of antibiotic No. 6270 and echinomycin in complexes with DNA increased 3--4 times as compared to the toxicity of the same antibiotics used without the complex. Under the experimental conditions with 3-fold intravenous administration at 72-hour intervals in doses equivalent by their acute toxicity, the antitumor activity of the echinomycin complex with DNA against the solid form of lymphosarcoma L10-1 was approximately 4 times lower than the activity of the antibiotic used alone. Like echinomycin, antibiotic No. 6270 in complex with DNA used according to the same administration scheme in doses equivalent by their acute toxicity had a lower inhibitory effect on growth of lymphosarcoma L10-1 and sarcoma 180 as compared to its use alone.     

Chemical probes reveal no evidence of Hoogsteen base pairing in complexes formed between echinomycin and DNA in solution

Five different DNA fragments have been treated with a range of conformationally sensitive reagents in an effort to probe structural changes in DNA associated with binding of the bis-intercalating antibiotic echinomycin. For each probe, the intensity and pattern of its reactivity with DNA have been analyzed in order to elucidate the effect of antibiotic binding on the accessibility of a specific site or sites to chemical attack. It was found that in one of the DNA fragments, pTyr2 DNA, several purine residues exhibit enhanced reactivity to diethyl pyrocarbonate (DEPC) in the absence of bound antibiotic, and that this strongly sequence specific reaction is enhanced in the presence of quite low echinomycin concentrations. The echinomycin-dependent reactivities towards DEPC of three homologous DNA fragments, chosen for their subtly different antibiotic binding characteristics, were also investigated. It was found that small changes in base sequence generate striking changes in susceptibility to modification by DEPC. The abolition of one antibiotic binding site leads to the creation of a new, intense DEPC-reactive site. In the presence of moderate concentrations of echinomycin, specific thymidine residues exhibit enhanced reactivity towards osmium tetroxide. No differences in the reactivities of the DNA fragments towards bromoacetaldehyde, S1 nuclease, dimethyl sulphate or potassium tetrachloropalladinate were observed in the presence of the antibiotic. DEPC reactions were performed on tubercidin (7-deaza-adenosine) to determine the DEPC reactive positions in situation where N-7 is inaccessible. Tubercidin was found to be generally resistant to attack by DEPC followed by treatment with base. We conclude that the bulk of structural changes induced by the binding of echinomycin to DNA do not involve Hoogsteen base pairing, but rather are due to sequence-specific unwinding of the helix in a manner which is strongly dependent on the nature of surrounding nucleotide sequences.     

Atomic force microscopy study of the structural effects induced by echinomycin binding to DNA

Atomic force microscopy (AFM) has been used to examine the conformational effects of echinomycin, a DNA bis-intercalating antibiotic, on linear and circular DNA. Four different 398 bp DNA fragments were synthesized, comprising a combination of normal and/or modified bases including 2,6-diaminopurine and inosine (which are the corresponding analogues of adenine and guanosine in which the 2-amino group that is crucial for echinomycin binding has been added or removed, respectively). Analysis of AFM images provided contour lengths, which were used as a direct measure of bis-intercalation. About 66 echinomycin molecules are able to bind to each fragment, corresponding to a site size of six base-pairs. The presence of base-modified nucleotides affects DNA conformation, as determined by the helical rise per base-pair. At the same time, the values obtained for the dissociation constant correlate with the types of preferred binding site available among the different DNA fragments; echinomycin binds to TpD sites much more tightly than to CpG sites. The structural perturbations induced when echinomycin binds to closed circular duplex pBR322 DNA were also investigated and a method for quantification of the structural changes is presented. In the presence of increasing echinomycin concentration, the plasmid can be seen to proceed through a series of transitions in which its supercoiling decreases, relaxes, and then increases.     

Structural isomers of bis-PNA bound to a target in duplex DNA

Upon binding of a decamer bis-PNA (H-Lys-TTCCTCTCTT-(eg1)(3)-TTCTCTCCTT-LysNH(2)) to a complementary target in a double-stranded DNA fragment, three distinct complexes were detected by gel mobility shift analysis. Using in situ chemical probing techniques (KMnO(4) and DMS) it was found that all three complexes represent bona fide sequence-specific PNA binding to the designated target, but the complexes were structurally different. One complex that preferentially formed at higher PNA concentrations contains two bis-PNA molecules per DNA target, whereas the other two complexes are genuine triplex invasion clamped structures. However, these two latter complexes differ by the path relative to the DNA target of the flexible ethylene-glycol linker connecting the two PNA oligomers that comprise a bis-PNA. We distinguish between one in which the linker wraps around the non-target DNA strand, thus making this strand part of the triplex invasion complex and another complex that encompass the target strand only. The implications of these results are discussed in terms of DNA targeting by synthetic ligands.