Dynamic light scattering and fluorescence study of the interaction between double-stranded DNA and poly(amido amine) dendrimers

The interaction between a cationic poly(amido amine) (PAMAM) dendrimer of generation 4 and double-stranded salmon sperm DNA in 10 mM NaBr solution has been investigated using dynamic light scattering (DLS) and steady-state fluorescence spectroscopy. The structural parameters of the formed aggregates as well as the complex formation process were studied in dilute solutions. When DNA is mixed with PAMAM dendrimers, it undergoes a transition from a semiflexible coil to a more compact conformation due to the electrostatic interaction present between the cationic dendrimer and the anionic polyelectrolyte. The DLS results reveal that one salmon sperm DNA molecule forms a discrete aggregate in dilute solution with several PAMAM dendrimers with a mean apparent hydrodynamic radius of 50 nm. These discrete complexes coexist with free DNA at low molar ratios of dendrimer to DNA, which shows that cooperativity is present in the complex formation. The formation of the complexes was confirmed by agarose gel electrophoresis measurements. DNA in the complexes was also found to be significantly more protected against DNase catalyzed digestion compared to free DNA. The number of dendrimers per DNA chain in the complexes was found to be approximately 35 as determined by steady-state fluorescence spectroscopy.     

Effect of polymer ionization on the interaction with DNA in nonviral gene delivery systems

The optimization of DNA-cationic polymer complexation is crucial for nonviral gene delivery. Although physicochemical characterization of the interaction between DNA and cationic polymers has recently attracted more attention in the nonviral DNA delivery field, the literature on the effect of varying polycation charge density on DNA-cationic polymer complexation is still scarce. Thus, the aim of this study was to systematically assess the influence of the degree of ionization of a weak cationic polyelectrolyte (poly[2-(dimethylamino)ethyl methacrylate] or DMAEMA homopolymer) on its ability to form complexes with DNA. This was achieved by varying the solution pH from 4.0 to 8.0 and analyzing the resulting effects on the binding affinity, thermodynamic properties, complex size, and morphology. Lowering the solution pH led to higher degrees of ionization for the cationic polymer and hence greater binding affinities with DNA, as judged by the increased propensity of the former to displace ethidium bromide from DNA and also by relatively low monomer:nucleotide molar ratio (0.8:1) required to retard the migration of free DNA. Isothermal titration microcalorimetry studies further confirmed that a stronger interaction occurred at low pH than at high pH. By decreasing the pH from 8.0 to 6.6, K(obs) increased from 7.8 x 10(5) to 20.4 x 10(5) M(-1). More efficient condensation at low pH was demonstrated by the reduction of ethidium bromide fluorescence in the loading wells from gel electrophoresis, decreased complex sizes without agglomeration occurring at high polymer/DNA ratios, together with discrete and dense spherical complexes observed in TEM studies. This may be attributed to the presence of electrostatic stabilization from excess cationic polymer chains, which provide a repulsive shell around the polymer/DNA complex. The physicochemical data indicate that the increased degree of ionization for the DMAEMA homopolymer at lower pH results in higher binding affinity, smaller and more compact complexes, and more efficient condensation. These findings therefore highlight the importance of the degree of ionization on DNA complex formation for weak cationic polyelectrolytes.     

Interaction of cationic surfactants with DNA: a single-molecule study

The interaction of cationic surfactants with single dsDNA molecules has been studied using force-measuring optical tweezers. For hydrophobic chains of length 12 and greater, pulling experiments show characteristic features (e.g. hysteresis between the pulling and relaxation curves, force-plateau along the force curves), typical of a condensed phase (compaction of a long DNA into a micron-sized particle). Depending on the length of the hydrophobic chain of the surfactant, we observe different mechanical behaviours of the complex (DNA-surfactants), which provide evidence for different binding modes. Taken together, our measurements suggest that short-chain surfactants, which do not induce any condensation, could lie down on the DNA surface and directly interact with the DNA grooves through hydrophobic–hydrophobic interactions. In contrast, long-chain surfactants could have their aliphatic tails pointing away from the DNA surface, which could promote inter-molecular interactions between hydrophobic chains and subsequently favour DNA condensation.     

Synthesis and biological activity of carbamate-linked cationic lipids for gene delivery in vitro

We have introduced a convenient synthesis method for carbamate-linked cationic lipids. Two cationic lipids N-[1-(2,3-didodecylcarbamoyloxy)propyl]-N,N,N-trimethylammonium iodide (DDCTMA) and N-[1-(2,3-didodecyl carbamoyloxy)propyl]-N-ethyl-N,N-dimethylammonium iodide (DDCEDMA), with identical length of hydrocarbon chains, alternative quaternary ammonium heads, carbamate linkages between hydrocarbon chains and quaternary ammonium heads, were synthesized for liposome-mediated gene delivery. Liposomes composed of DDCEDMA and DOPE in 1:1 ratio exhibited a lower zeta potential as compared to those made of pure DDCEDMA alone, which influences their DNA-binding ability. pGFP-N2 plasmid was transferred by cationic liposomes formed from the above cationic lipids into Hela and Hep-2 cells, and the transfection efficiency of some of cationic liposomes was superior or parallel to that of two commercial transfection agents, Lipofectamine2000 and DOTAP. Combined with the results of the agarose gel electrophoresis and transfection experiment, the DNA-binding ability of cationic lipids was too strong to release DNA from complex in the transfection, which could lead to relative low transfection efficiency and high cytotoxicity.     

Oligonucleotides form a duplex with non-helical properties on a positively charged surface

The double helix is known to form as a result of hybridization of complementary nucleic acid strands in aqueous solution. In the helix the negatively charged phosphate groups of each nucleic acid strand are distributed helically on the outside of the duplex and are available for interaction with cationic groups. Cation-coated glass surfaces are now widely used in biotechnology, especially for covalent attachment of cDNAs and oligonucleotides as surface-bound probes on microarrays. These cationic surfaces can bind the nucleic acid backbone electrostatically through the phosphate moiety. Here we describe a simple method to fabricate DNA microarrays based upon adsorptive rather than covalent attachment of oligonucleotides to a positively charged surface. We show that such adsorbed oligonucleotide probes form a densely packed monolayer, which retains capacity for base pair-specific hybridization with a solution state DNA target strand to form the duplex. However, both strand dissociation kinetics and the rate of DNase digestion suggest, on symmetry grounds, that the target DNA binds to such adsorbed oligonucleotides to form a highly asymmetrical and unwound duplex. Thus, it is suggested that, at least on a charged surface, a non-helical DNA duplex can be the preferred structural isomer under standard biochemical conditions.     

Properties of Bacteriophage T4 ribonucleoside diphosphate reductase subunits coded by nrdA and nrdB mutants

As a part of the study of the bacteriophage T4-induced deoxyribonucleotide synthetase complex, an investigation has been made of the T4 ribonucleoside diphosphate reductases formed by a series of mutants of nrdA and B, the genes coding, respectively, for the alpha 2 and beta 2 subunits of the enzyme. dATP affinity columns were used to isolate the enzyme by a single-step procedure. The molecular weights of the alpha and beta chains have been found to be 84,000 and 43,500, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since alpha 2 beta 2 is bound to dATP affinity columns through allosteric effector sites on alpha 2, it is possible to monitor the binding of beta 2 to alpha 2. dTTP- and ATP-Sepharose columns did not bind T4 alpha 2 beta 2, although the corresponding nucleoside triphosphates are effectors of the enzyme and although the alpha 2 subunit of the host enzyme binds to these columns. Missense mutants of nrdA and B forming alpha 2 and beta 2 subunits that lacked catalytic activity but retained the ability to form the alpha 2 beta 2 complex have been described. The 50,000-dalton fragment formed by an amber mutant of nrdA did not bind to the dATP affinity column, providing evidence that a region of the carboxyl-terminal segment of the alpha chain is required for retention. The beta 2 subunit appears to protect the alpha 2 protein. On infection by nrdB mutants not forming beta 2, the alpha protein chain was cleaved specifically to form 3 protein chains of 61,000, 57,000, and 24,500 daltons, which retain the ability to bind to dATP-Sepharose. Some effects of mutation on the interaction of the alpha and beta chains of the enzyme with the deoxyribonucleotide synthetase complex have been examined.     

Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis

Three proteins catalyze RNA-primed DNA synthesis on the lagging strand side of the replication fork of bacteriophage T7. Oligoribonucleotides are synthesized by T7 gene 4 protein, which also provides helicase activity. DNA synthesis is catalyzed by gene 5 protein of the phage, and processivity of DNA synthesis is conferred by Escherichia coli thioredoxin, a protein that is tightly associated with gene 5 protein. T7 DNA polymerase and gene 4 protein associate to form a complex that can be isolated by filtration through a molecular sieve. The complex is stable in 50 mM NaCl but is dissociated by 100 mM NaCl, a salt concentration that does not inhibit RNA-primed DNA synthesis. T7 DNA polymerase forms a stable complex with single-stranded M13 DNA at 50 mM NaCl as measured by gel filtration, and this complex requires 200 mM NaCl for dissociation, a salt concentration that inhibits RNA-primed DNA synthesis. Gene 4 protein alone does not bind to single-stranded DNA. In the presence of MgCl2 and dTTP or beta, gamma-methylene dTTP, a gene 4 protein-M13 DNA complex that is stable at 200 mM NaCl is formed. The affinity of DNA polymerase for both gene 4 protein and single-stranded DNA leads to the formation of a gene 4 protein-DNA polymerase-M13 DNA complex even in the absence of nucleoside triphosphates. However, the binding of each protein to DNA plays an important role in mediating the interaction of the proteins with each other. High concentrations of single-stranded DNA inhibit RNA-primed DNA synthesis by diluting the amount of proteins bound to each template and reducing the frequency of protein-protein interactions. Preincubation of gene 4 protein, DNA polymerase, and M13 DNA in the presence of dTTP forms protein-DNA complexes that most efficiently catalyze RNA-primed DNA synthesis in the presence of excess single-stranded competitor DNA.     

Models of bleomycin interactions with poly(deoxyadenylylthymidylic acid). Fluorescence and proton nuclear magnetic resonance studies of cationic thiazole amides related to bleomycin A2

The interaction of eight 2-substituted thiazole-4-carboxamides, structurally related to cationic terminus of bleomycin A2, with poly(deoxyadenylylthymidylic acid) [poly(dA-dT)] has been studied by using proton nuclear magnetic resonance and fluorescence spectroscopy. These analogues have been used as probes of the complex formed between the parent drug molecule and poly(dA-dT). Aliphatic substituents on the 2' position of 2,4'-bithiazole derivatives restrict the ability of the aromatic ring system to intercalate in the double-helical form of the polynucleotide. Absence or partial removal of the 2' substituent enhances intercalation of the bithiazole system. The cationic side chain does not appear to be involved in the stabilization of any of these complexes, although it may be necessary for their formation. A 2,4':2',4"-terthiazole derivative shows a substantial degree of intercalation which is accompanied by extensive immobilization of the cationic side chain. This suggests that insertion of the aromatic system into the nucleic acid causes the cationic side chain to be pulled in also. Monothiazole analogues do not appear to bind, indicating that at least two thiazole rings are necessary for binding or that proper spacing between the two side chains on either side of the thiazole system is important for binding. The relation of the interactions of these analogues to the biochemical and biological properties of the parent bleomycins is discussed as is the possible use of these data in the design of synthetic bleomycin derivatives having varying affinities and specificities for DNA.     

Characterization of DNA primase complex isolated from the archaeon, Thermococcus kodakaraensis

In most organisms, DNA replication is initiated by DNA primases, which synthesize primers that are elongated by DNA polymerases. In this study, we describe the isolation and biochemical characterization of the DNA primase complex and its subunits from the archaeon Thermococcus kodakaraensis. The T. kodakaraensis DNA primase complex is a heterodimer containing stoichiometric levels of the p41 and p46 subunits. The catalytic activity of the complex resides within the p41 subunit. We show that the complex supports both DNA and RNA synthesis, whereas the p41 subunit alone marginally produces RNA and synthesizes DNA chains that are longer than those formed by the complex. We report that the T. kodakaraensis primase complex preferentially interacts with dNTP rather than ribonucleoside triphosphates and initiates RNA as well as DNA chains de novo. The latter findings indicate that the archaeal primase complex, in contrast to the eukaryote homolog, can initiate DNA chain synthesis in the absence of ribonucleoside triphosphates. DNA primers formed by the archaeal complex can be elongated extensively by the T. kodakaraensis DNA polymerase (Pol) B, whereas DNA primers formed by the p41 catalytic subunit alone were not. Supplementation of reactions containing the p41 subunit with the p46 subunit leads to PolB-catalyzed DNA synthesis. We also established a rolling circle reaction using a primed 200-nucleotide circle as the substrate. In the presence of the T. kodakaraensis minichromosome maintenance (MCM) 3' → 5' DNA helicase, PolB, replication factor C, and proliferating cell nuclear antigen, long leading strands (>10 kb) are produced. Supplementation of such reactions with the DNA primase complex supported lagging strand formation as well.     

Surfactant-DNA gel particles: formation and release characteristics

Aqueous mixtures of oppositely charged polyelectrolytes undergo associative phase separation, resulting in coacervation, gelation, or precipitation. This phenomenon has been exploited here to form DNA gel particles by interfacial diffusion. We report on the formation of DNA gel particles by mixing solutions of DNA (either single-stranded (ssDNA) or double-stranded (dsDNA)) with solutions of cationic surfactant cetyltrimetrylammonium bromide (CTAB). By using CTAB, the formation of DNA reservoir gel particles, without adding any kind of cross-linker or organic solvent, has been demonstrated. Particles have been characterized with respect to the degree of DNA entrapment, surface morphology, and secondary structure of DNA in the particles. The swelling/deswelling behavior and the DNA release have been investigated in response to salt additions. Analysis of the data has suggested a higher degree of interaction between ssDNA and the cationic surfactant, confirming the stronger amphiphilic character of the denatured DNA. Fluorescence microscopy studies have suggested that the formation of these particles is associated with a conservation of the secondary structure of DNA.     

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.     

Molecular Dynamics Simulations of DNA-Polycation Complex Formation

Complexes formed from DNA and polycations are of interest because of their potential use in gene therapy; however, there remains a lack of understanding of the structure and formation of DNA-polycation complexes at atomic scale. In this work, molecular dynamics simulations of the DNA duplex d(CGCGAATTCGCG) in the presence of polycation chains are carried out to shed light on the specific atomic interaction that result in complex formation. The structures of complexes formed from DNA with polyethylenimine, which is considered one of the most promising DNA vector candidates, and a second polycation, poly-L-lysine, are compared. After an initial separation of ∼50 Å, the DNA and polycation come together and form a stable complex within 10 ns. The DNA does not undergo any major structural changes on complexation and remains in the B-form. In the formed complex, the charged amine groups of the polycation mainly interact with DNA phosphate groups, with polycation intrusion into the major and minor grooves dependent on the identity and charge state of the polycation. The ability of the polycation to effectively neutralize the charge of the DNA phosphate groups and the resulting influence on the DNA helix interaction are discussed.     

Isolation and characterization of precursors in bacteriophage T4 baseplate assembly. II. Purification of the protein products of genes 10 and 11 and the in vitro formation of the P(10/11) complex

Two bacteriophage T4 proteins which are precursors to the phage baseplate have been purified to homogeneity. These proteins, P10 and P11, are components of the P(10/11) complex, which is the first intermediate in the assembly of T4 baseplate 1/6th arms. Each protein was isolated from cells infected with a T4 amber mutant defective in the production of the other protein. Thus these purified proteins have never been assembled into the P(10/11) complex in vivo. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and the ability of these proteins to block the phage killing activity of specific antisera were used to monitor the purification steps. Sedimentation equilibrium experiments reveal a molecular weight of 188,000 g/mol for P10 and 60,000 g/mol for P11. These data together with the previously determined molecular weights of the gene 10 and gene 11 polypeptide chains (King & Mykolajewycz, 1973) and the in vivo assembled P(10/11) complex (Berget & King, 1978b) are consistent with P10 being a dimer of the product of gene 10, P11 being a dimer of the product of gene 11, and P(10/11) being a tetramer containing one of each of these dimers. Purified P10 and P11 are active in assembly because they complement 10- and 11- defective extracts, respectively, to form viable bacteriophage in vitro. Furthermore, these proteins assemble in vitro to form a protein structure identical to the P(10/11) complex formed in vivo as determined by non-denaturing gel electrophoresis. This P(10/11) complex formed in vitro complements 10-/11- defective extracts to form viable phage. The overall extent of this in vitro assembly reaction is not affected by NaCl to 1.5 M or 2% Triton X-100. The reaction is, however, prevented by the denaturing effects of urea and sodium dodecyl sulfate.     

Stabilization of triple-stranded oligonucleotide complexes: use of probes containing alternating phosphodiester and stereo-uniform cationic phosphoramidate linkages.

Pyrimidine oligonucleotides containing alternating anionic and stereo-uniform cationic N-(dimethylamino-propyl)phosphoramidate linkages [e.g. d(T+T-)7T, d(T+T-)2(T+C-)5T and (U'+U'-)7dT, where U' is 2'-O-methyluridine)] are shown to bind to complementary double-stranded DNA segments in 0.1 M NaCl at pH 7 to form triple-stranded complexes with the pyrimidine.purine.pyrimidine motif. For each of the sequences investigated, one stereoisomer bound with higher affinity, and the other stereoisomer with lower affinity, than the corresponding all-phosphodiester oligonucleotide. The stereoisomer of d(T+T-)7T that interacted weakly with a dT.dA target in 0.1 M NaCl formed a novel dA.dA.dT triple-stranded complex with poly(dA) or d(Al5C4A15) in 1 M NaCl; in contrast, the stereoisomer that bound strongly to the dT.dA target failed to form a dA.dA.dT triple-stranded complex.     

Voltammetric and spectroscopic studies on methyl green and cationic lipid bound to calf thymus DNA

DNA interaction with cationic lipids promises to be a versatile and effective synthetic transfection agent. This paper presents the study on binding of a simple artificial cationic lipid, cetyltrimethylammonium bromide (CTAB), to calf thymus DNA (CT DNA) prior to the condensation process, taking methyl green (MG) as a probe. The results show that the CTAB binds to DNA through electrostatic interaction forming a hydrophobic complex, thus changing the micro-environment of duplex of DNA, so the binding state of MG and DNA is changed, and a complex CTAB-CT DNA-MG is formed. This fact suggests a new way to mediate the conformation of molecular assemblies of DNA and lipids.     

Model of the packing of DNA and histone octamer in the structure of the nucleosome

A model is proposed which describes the packing of polypeptide chains of histone molecules in the octamer (H3--H4--H2A--H2B)2, and interlocation of DNA and octamer in the nucleosome. DNA packing in the nucleosome is provided for by electrostatic interactions between DNA phosphates and cationic groups located on the globular part surface of histones octamer. The cationic groups of N- and C-end regions of the histone molecules (histones H3 and H4 in particular) additionally stabilize the nucleosome structure.     

Branched structures in the intracellular DNA of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) replication produces large intracellular DNA molecules that appear to be in a head-to-tail concatemeric arrangement. We have previously suggested (A. Severini, A.R. Morgan, D.R. Tovell, and D.L.J. Tyrrell, Virology 200:428-435, 1994) that these DNA species may have a complex branched structure. We now provide direct evidence for the presence of branches in the high-molecular-weight DNA produced during HSV-1 replication. On neutral agarose two-dimensional gel electrophoresis, a technique that allows separation of branched restriction fragments from linear fragments, intracellular HSV-1 DNA produces arches characteristic of Y junctions (such as replication forks) and X junctions (such as merging replication forks or recombination intermediates). Branched structures were resolved by T7 phage endonuclease I (gene 3 endonuclease), an enzyme that specifically linearizes Y and X structures. Resolution was detected by the disappearance of the arches on two-dimensional gel electrophoresis. Branched structures were also visualized by electron microscopy. Molecules with a single Y junction were observed, as well as large tangles containing two or more consecutive Y junctions. We had previously shown that a restriction enzyme which cuts the HSV-1 genome once does not resolve the large structure of HSV-1 intracellular DNA on pulsed-field gel electrophoresis. We have confirmed that result by using sucrose gradient sedimentation, in which both undigested and digested replicative intermediates sediment to the bottom of the gradient. Taken together, our experiments show that the intracellular HSV-1 DNA is held together in a large complex by frequent branches that create a network of replicating molecules. The fact that most of these branches are Y structures suggests that the network is held together by frequent replication forks and that it resembles the replicative intermediates of bacteriophage T4. Our findings add complexity to the simple model of rolling-circle DNA replication, and they pose interesting questions as to how the network is formed and how it is resolved for packaging into progeny virions.     

Biophysical characterization of quaternary pyridinium functionalized polynorbornenes for DNA complexation and their cellular interactions

Cationic polymers with hydrophobic side chains have gained great interest as DNA carriers since they form a compact complex with negatively charged DNA phosphate groups and interact with the cell membrane. Amphiphilic polyoxanorbornenes with different quaternary alkyl pyridinium side chains with ethyl‐p(OPy2) and hexyl units‐p(OPy6) bearing 10 kDa MWT were synthesized by living Ring‐Opening Metathesis Polymerization method. The physicochemical characteristics: critical micellar concentration, size distribution, surface charge, and condensation of polymer/DNA complex were investigated. Morphology of complexes was monitored by Atomic force microscopy. Cytotoxicity and interaction of these complexes with model lipid vesicles mimicking the cell membrane were examined. These polymers were enabled to form small sized complexes of DNA, which interact with model membrane vesicles. It was found that the nature of hydrophobicity of the homopolymers significantly impacts rates of DNA complexation and the surface charge of the resulting complexes. These results highlight the prospect of the further examinations of these polymers as gene carriers.     

The cohering telomeres of Oxytricha.

We have studied the process by which purified Oxytricha macronuclear DNA associates with itself to form large aggregates. The various macronuclear DNA molecules all have the same terminal or telomeric DNA sequences that are shown below. 5' C4A4C4A4C4--mean length----G4T4G4T4G4T4G4T4G4 G4T4G4T4G4T4G4T4G4-----2.4 kb------C4A4C4A4C4. When incubated at high concentrations, these telomeric sequences cohere with one another to form an unusual structure--one that is quite different from any DNA structure so far described. The evidence for this is the following: 1) These sequences cohere albeit slowly, in the presence of relatively high concentrations of Na+, and no other cation tested. This contrasts with the rapid coherence of complementary single-chain terminals of normal DNA (sticky ends) which occurs in the presence of any cation tested. 2) If the cohered form is transferred into buffers containing a special cation, K+, it becomes much more resistant to dissociation by heating. We estimate that K+ increases the thermal stability by 25 degrees or more. The only precedent known (to us) for a cation-specific stabilization is that seen in the quadruplex structure formed by poly I. The thermal stability of double helical macronuclear DNA depends on the cation concentration, but not the cation type. Limited treatment with specific nucleases show that the 3' and 5'-ended strands are essential for the formation of the cohering structure. Once in the cohered form, the telomeric sequences are protected from the action of nucleases. Coherence is inhibited by specific, but not by non-specific, synthetic oligomers, and by short telomeric fragments with or without their terminal single chains. We conclude that the coherence occurs by the formation of a novel condensed structure that involves the terminal nucleotides in three or four chains.