Deciphering the energy landscape of the interaction uranyl-DCP with antibodies using dynamic force spectroscopy

Previous studies on molecular recognition of uranyl-DCP (dicarboxy-phenanthroline chelator) compound by two distinct monoclonal antibodies (Mabs U04S and U08S) clearly showed the presence of a biphasic shape in Bell-Evans’ plots and an accentuated difference in slopes at the high loading rates. To further explore the basis in the slope difference, we have performed complementary experiments using antibody PHE03S, raised against uranyl-DCP but, presenting a strong cross-reactivity toward the DCP chelator. This work allowed us to obtain a reallocation of the respective contributions of the metal ion itself and that of the chelator. Results led us to propose a 2D schematic model representing two energy barriers observed in the systems Mabs U04S- and U08S-[UO₂-DCP] where the outer barrier characterizes the interaction between UO₂ and Mab whereas the inner barrier characterizes the interaction between DCP and Mab. Using dynamic force spectroscopy, it is thus possible to dissect molecular interactions during the unbinding between proteins and ligands.     

Energy landscape of chelated uranyl: antibody interactions by dynamic force spectroscopy

We used dynamic force spectroscopy (DFS) to explore the energy landscape of interactions between a chelated uranyl compound and a monoclonal antibody raised against the uranyl-dicarboxy-phenanthroline complex. We estimated the potential energy barrier widths and the relevant thermodynamic rate constants along the dissociation coordinate. Using atomic force microscopy, four different experimental setups with or without the uranyl ion in the chelate ligand, we have distinguished specific and nonspecific binding in the binding affinity of the uranyl compound to the antibody. The force loading rates for our system were measured from 15 to 26,400 pN/s. The results showed two regimes in the plot of the most probable unbinding force versus the logarithm of the loading rate, revealing the presence of two (at least) activation barriers. Analyses of DFS suggest parallel multivalent binding present in either regime. We have also built a molecular model for the variable fragment of the antibody and used computational graphics to dock the chelated uranyl ion into the binding pocket. The structural analysis led us to hypothesize that the two regimes originate from two interaction modes: the first one corresponds to an energy barrier with a very narrow width of 0.5 +/- 0.2 A, inferring dissociation of the uranyl ion from its first coordination shell (Asp residue); the second one with a broader energy barrier width (3.9 +/- 0.3 A) infers the entire chelate compound dissociated from the antibody. Our study highlights the sensitivity of DFS experiments to dissect protein-metal compound interactions.     

Novel monoclonal antibodies with specificity for chelated uranium(VI): isolation and binding properties

A derivative of 1,10-phenanthroline that binds to UO(2)(2+) with nanomolar affinity was found to be a very effective immunogen for the generation of antibodies directed toward chelated complexes of hexavalent uranium. This study describes the synthesis of 5-isothiocyanato-1,10-phenanthroline-2,9-dicarboxylic acid and its use in the generation and functional characterization of a group of monoclonal antibodies that recognize the most soluble and toxic form of uranium, the hexavalent uranyl ion (UO(2)(2+)). Three different monoclonal antibodies (8A11, 10A3, and 12F6) that recognize the 1:1 complex between UO(2)(2+) and 2,9-dicarboxy-1,10-phenanthroline (DCP) were produced by the injection of BALB/c mice with DCP-UO(2)(2+) covalently coupled to a carrier protein. Equilibrium dissociation constants for the binding of DCP-UO(2)(2+) to antibodies 8A11, 10A3, and 12F6 were 5.5, 2.4, and 0.9 nM, respectively. All three antibodies bound the metal-free DCP with roughly 1000-fold lower affinity. The second-order rate constants for the bimolecular association of each antibody with soluble DCP-UO(2)(2+) were in the range of 1 to 2 x 10(7) M(-1) s(-1). Binding studies conducted with structurally related chelators and 21 metal ions demonstrated that each of these three antibodies was highly specific for the soluble DCP-UO(2)(2+) complex. Detailed equilibrium binding studies conducted with three other derivatives of DCP, either complexed with UO(2)(2+) or metal-free, suggested that the antigen binding sites on the three antibodies have significant functional and structural similarities. Biomolecules that bind specifically to uranium will be at the heart of any new biotechnology developed to monitor and control uranium contamination. The three antibodies described herein possess sufficient affinity and specificity to support the development of immunoassays for hexavalent uranium in environmental and clinical samples.     

Altered antigenic disposition of peroxisomal urate oxidase in PEX5-defective Chinese hamster ovary cells

Since Chinese hamster ovary (CHO) cells never express urate oxidase (UO), we tried to establish cell lines stably producing UO in order to elucidate the peroxisomal import process. The enzyme is a peroxisome targeting signal 1 (PTS1) protein harboring SKL motif at the carboxy-terminus [Biochem. Biophys. Res. Commun. 158 (1989) 991] and PEX5 protein (Pex5p) is supposed to be involved in the import process [Nat. Genet. 9 (1995) 115; J. Cell Biol. 130 (1995) 51]. We transfected a cDNA encoding rat UO into both wild type and PEX5-defective CHO cells to isolate each cell line stably producing the enzyme. While we examined the import process of UO in mutant cells, we noticed an interesting observation by using polyclonal antibody U1 or U2, which separately recognizes epitopes of UO. U1 antibody mainly interacts with epitopes in the amino-terminal region of UO. On the other hand, U2 antibody reacts with many epitopes distributed in the broad region of UO molecule. When UO produced in cultured cells was stained with U2 antibody, the enzyme was detected in peroxisomes of both wild type and PEX5-mutant cells. Whereas, U1 antibody stained the peroxisomal UO in wild type cells, but not in PEX5-mutant cells. These immunocytochemical observations suggest that the epitopes at the amino-terminal region of UO will be concealed in mutant cells. When the mutant cells were transfected with wild type PEX5 cDNA, U1 antibody came to react with UO in peroxisomes of mutant cells. The restoration indicates that the exposure of N-terminal epitopes of UO will depend upon the functional Pex5p. Immunoelectron microscopic observation showed that the peroxisomal import of UO was partially retarded in PEX5 mutant cells. The observation also supported the fact that UO was mainly localized in the peroxisomal matrix of wild type cells but in the membrane of mutant cells.     

Ion exchange/complexation of the uranyl ion by Rhizopus biosorbent

Nonliving biomass of nine Rhizopus species effectively sequestered the uranyl ion from solution, taking up 150-250 mg U/g dry cells at 300 ppm U equilibrium concentration in solution, and 100-160 mg U/g dry cells with 100 ppm U in solution. The affinity of this biosorbent for the uranyl ion was found to be affected by timing of harvesting and medium composition. Uptake of the uranyl ion by nonliving biomass of Rhizopus oligosporus was due to ion exchange or complexation, since binding was reversed by the addition of complexing ligands or the reduction of pH to a value less than 2. Uptake isotherms were interpreted in terms of a model of multiple equilibria. At pH 相似文献   

Accumulation of uranium at low concentration by the green alga Scenedesmus obliquus 34

Accumulation of UO ₂ ^{2 +} by Scenedesmus obliquus 34 was rapid and energy-independent and the biosorption of UO ₂ ^{2 +} could be described by the Freundlich adsorption isotherm below the maximum adsorption capacity (75 mg g-¹ dry wt). The optimum pH for uranium uptake was between 5.0_8.5.0.1_2.0 M NaCl enhanced uranyl, while Cu²⁺, Ni²⁺, Zn²⁺, Cd²⁺ and Mn²⁺ competed slightly with uranyl. Pretreatment had an unexpected effect on biosorption. After being killed by 0.1 M HCl, S. Obliquus 34 showed 45% of the uptake capacity of the control in which fresh cells were suspended directly in uranyl solution, while the pretreatment of cells by 0.1 M NaOH, 2.0 M NaCl, ethanol or heating decreased uptake slightly. Fresh S. obliquus 34 at 1.2_2.4 mg dry wt mL-1 was able to decrease U from 5.0 to 0.05 mg L-1 after 4_6 equilibrium stages with batch adsorption. Deposited U could be desorbed by pH 4.0 buffer. It is suggested that U was captured by effective groups or by capillary action in the cell wall in the form of [UO2OH]+. This revised version was published online in August 2006 with corrections to the Cover Date.     

Force history dependence of receptor-ligand dissociation

Receptor-ligand bonds that mediate cell adhesion are often subjected to forces that regulate their dissociation via modulating off-rates. Off-rates control how long receptor-ligand bonds last and how much force they withstand. One should therefore be able to determine off-rates from either bond lifetime or unbinding force measurements. However, substantial discrepancies exist between the force dependence of off-rates derived from the two types of measurements even for the same interactions, e.g., selectins dissociating from their ligands, which mediate the tethering and rolling of leukocytes on vascular surfaces during inflammation and immune surveillance. We used atomic force microscopy to measure survival times of P-selectin dissociating from P-selectin glycoprotein ligand 1 or from an antibody in both bond lifetime and unbinding force experiments. By a new method of data analysis, we showed that the discrepancies resulted from the assumption that off-rates were functions of force only. The off-rates derived from forced dissociation data depended not only on force but also on the history of force application. This finding provides a new paradigm for understanding how force regulates receptor-ligand interactions.     

Change of the unbinding mechanism upon a mutation: a molecular dynamics study of an antibody-hapten complex

We study forced unbinding of fluorescein from the wild type (WT) and a mutant [H(H58)A] of the single-chain variable-fragment (scFv) anti-fluorescein antibody FITC-E2 by molecular dynamics simulations using various pulling techniques. A large number of long simulations were needed to obtain statistically meaningful results as both the wild type and the H(H58)A mutant unbinding occurs through multiple pathways, often with metastable intermediates. For the wild type, the rate-limiting step in the unbinding process corresponds to the breaking of the non-native interactions characteristic of a specific intermediate. The H(H58)A mutation disfavors the occurrence of this intermediate. Two events where the hapten partially unbinds in the absence of pulling force are observed in extensive equilibrium simulations of the wild type, and their analysis indicates that forced unbinding and spontaneous unbinding proceed along similar pathways. The different unbinding mechanisms observed in the simulations suggest a possible reason for the difference in the experimental off-rate between the two antibodies. We predict mutations that are expected to modulate the occurrence of the unbinding intermediate. For two such new mutants [H(H58)A and S(H52)A], our predictions are validated in silico by additional simulations. The accompanying paper in this issue by Honegger et al. reports the X-ray structure of FITC-E2 with a derivative of fluorescein, which was used as the starting conformation for the work presented here.     

Dynamic force spectroscopy of the digoxigenin-antibody complex

Small ligands and their receptors are widely used non-covalent couplers in various biotech applications. One prominent example, the digoxigenin-antibody complex, was often used to immobilize samples for single molecule force measurements by optical trap or AFM. Here, we employed dynamic AFM spectroscopy to demonstrate that a single digoxigenin-antibody bond is likely to fail even under moderate loading rates. This effect potentially could lower the yield of measurements or even obscure the unbinding data of the sample by the rupture events of the coupler. Immobilization by multiple antibody-antigen bonds, therefore, is highly recommended. The analysis of our data revealed a pronounced loading rate dependence of the rupture force, which we analyzed based on the well-established Bell-Evans-model with two subsequent unbinding barriers. We could show that the first barrier has a width of Deltax(1)=1.15 nm and a spontaneous rate of k(off1)=0.015 s(-1) and the second has a width of Deltax(2)=0.35 nm and a spontaneous rate of k(off2)=4.56 s(-1). In the crossover region between the two regimes, we found a marked discrepancy between the predicted bond rupture probability density and the measured rupture force histograms, which we discuss as non-Markovian contribution to the unbinding process.     

Evaluation of temperature effect on the interaction between β-lactoglobulin and anti-β-lactoglobulin antibody by atomic force microscopy

Molecular recognition such as antigen-antibody interaction is characterized by the parameters of kinetics and the energy landscape. Examinations of molecules involved in the interaction at different temperatures using atomic force microscopy (AFM) can provide information on not only the effects of temperature on the unbinding force between a molecule of interest and a complementary molecule but also the parameters of kinetics and the energy landscape for dissociation of the molecular complex. We investigated the effect of temperature on the dissociation process of the complex of β-lactoglobulin and anti-bovine β-lactoglobulin IgG polyclonal antibody using AFM. Measurements of the unbinding forces between β-lactoglobulin and the antibody were performed at 25, 35, and 45 °C. The following results were obtained in our present study: (i) The unbinding forces decreased as temperature increased, suggesting that the binding force between β-lactoglobulin and the antibody includes the force originating from temperature-dependent interactions (e.g., hydrogen bonding). (ii) At each temperature, the unbinding force exhibited two linear regimes in the force spectra, indicating that the dissociation process of the β-lactoglobulin-antibody complex passes at least two energy barriers from the bound state to the dissociated state. (iii) The dissociation rates at zero force and the position of energy barriers increased as temperature increased. (iv) The heights of the two energy barriers in the reaction coordinates were 49.7 k(B)T and 14.5 k(B)T. (v) The values of roughness of the barriers were ca. 6.1 k(B)T and 3.2 k(B)T. Overall, the present study using AFM revealed more information about the β-lactoglobulin-antibody interaction than studies using conventional bulk measurement such as surface plasmon resonance.     

Uranium(VI) complexes with phospholipid model compounds--a laser spectroscopic study

We present the complex formation of the uranyl ion (UO(2)(2+)) in the aqueous system with phosphocholine, O-phosphoethanolamine and O-phosphoserine. These phosphonates (R-O-PO(3)(2-)) represent the hydrophilic head groups of phospholipids. The complexation was investigated by time-resolved laser-induced fluorescence spectroscopy (TRLFS) at pH=2-6. An increase of the fluorescence intensity, connected with a strong red-shift of about 8 nm compared to the free uranyl ion, indicates a complex formation between UO(2)(2+) and the phosphonates already at pH=2. Even at pH=6 these complexes prevail over the uranyl hydroxide and carbonate species, which are generated naturally at this pH. At pH=4 and higher a 1:2 complex between uranyl and O-phosphoserine was found. Complexes with a metal-to-ligand ratio of 1:1 were observed for all other ligands. Fluorescence lifetimes, emission maxima and complex stability constants at T=22+/-1 degrees C are reported. The TRLFS spectra of uranyl complexes with two phosphatidic acids (1,2-dimyristoyl-sn-glycero-3-phosphate and 1,2-dipalmitoyl-sn-glycero-3-phosphate), which represent the apolaric site of phospholipids, show in each case two different species.     

Steered molecular dynamics simulations on the "tail helix latch" hypothesis in the gelsolin activation process

The molecular basis of the "tail helix latch" hypothesis in the gelsolin activation process has been studied by using the steered molecular dynamics simulations. In the present nanosecond scale simulations, the tail helix of gelsolin was pulled away from the S2 binding surface, and the required forces were calculated, from which the properties of binding between the tail helix and S2 domain and their dynamic unbinding processes were obtained. The force profile provides a detailed rupture mechanism that includes six major unbinding steps. In particular, the hydrogen bonds formed between Arg-207 and Asp-744 and between Arg-221 and Leu-753 are of the most important interaction pairs. The two hydrogen bond "clamps" stabilize the complex. The subsequent simulation on Arg-207-Ala (R207A) mutation of gelsolin indicated that this mutation facilitates the unbinding of the tail helix and that the contribution of the hydrogen bond between Arg-207 and Asp-744 to the binding is more than 50%, which offers a new clue for further mutagenesis study on the activation mechanism of gelsolin. Surrounding water molecules enhance the stability of the tail helix and facilitate the rupture process. Additionally, temperature also has a significant effect on the conformation of the arginine and arginine-related interactions, which revealed the molecular basis of the temperature dependence in gelsolin activation.     

Forces and energetics of hapten-antibody dissociation: a biased molecular dynamics simulation study

The unbinding of fluorescein from the single-chain Fv fragment of the 4D5Flu antibody is investigated by biased molecular dynamics with an implicit solvation model. To obtain statistically meaningful results, a large number of unbinding trajectories are calculated; they involve a total simulation time of more than 200 ns. Simulations are carried out with a time-dependent perturbation and in the presence of a constant force. The two techniques, which provide complementary information, induce unbinding by favoring an increase in the distance between the ligand and the antibody. This distance is an appropriate progress variable for the dissociation reaction and permits direct comparison of the unbinding forces in the simulations with data from atomic force microscopy (AFM). The time-dependent perturbation generates unfolding pathways that are close to equilibrium and can be used to reconstruct the mean force; i.e. the derivative of the potential of mean force, along the reaction coordinate. This is supported by an analysis of the overall unbinding profile and the magnitude of the mean force, which are similar to those of the unbinding force (i.e. the external force due to the time-dependent perturbation) averaged over several unbinding events.The multiple simulations show that unbinding proceeds along a rather well-defined pathway for a broad range of effective pulling speeds. Initially, there is a distortion of the protein localized in the C-terminal region followed by the fluorescein exit from the binding site. This occurs in steps that involve breaking of specific electrostatic and van der Waals interactions. It appears that the simulations do not explore the same barriers as those measured in the AFM experiments because of the much higher unfolding speed in the former. The dependence of the force on the logarithm of the loading rate is linear and the slope is higher than in the AFM, in agreement with experiment in other systems, where different slopes were observed for different regimes. Based on the unbinding events, mutations in the 4D5Flu antigen binding site are predicted to result in significant changes in the unbinding force.     

Force measurement for antigen-antibody interaction by atomic force microscopy using a photograft-polymer spacer

To determine the intermolecular force on protein-protein interaction (PPI) by atomic force microscopy (AFM), a photograft-polymer spacer for protein molecules on both surfaces of the substrate and AFM probe tip was developed, and its effectiveness was assessed in a PPI model of a pair of human serum albumin (HSA) and its monoclonal antibody (anti-HSA). A carboxylated photoiniferter, N-(dithiocarboxy)sarcosine, was derivatized on both surfaces of the glass substrate and AFM probe tip, and subsequently water-soluble nonionic vinyl monomers, N,N-dimethylacrylamide (DMAAm), were graft-polymerized on them upon ultraviolet light irradiation. DMAAm-photograft-polymerized spacers with carboxyl groups at the growing chain end but with different chain lengths on both surfaces were prepared. The proteins were covalently bound to the carboxyl terminus of the photograft-polymer chain using a water-soluble condensation agent. The effects of the graft-spacer length on the profile of the force-distance curves and on the unbinding characteristics (unbinding force and unbinding distance) were examined in comparison with those in the case of the commercially available poly(ethylene glycol) (PEG) spacer. The frequency of the nonspecific adhesion force profile was markedly decreased with the use of the photograft spacers. Among the force curves detected, a high frequency of single-peak curves indicating the unbinding process of a single pair of proteins and a very low frequency of multiple-peak profiles were observed for the photograft spacers, regardless of the graft chain length, whereas a high frequency of no-force peaks was noted. These observations were in marked contrast with those for the PEG spacer. The force peak values determined ranged from 88 to 94 pN, irrespective of the type of spacer, while the standard deviation of force distribution observed for the photograft spacer was lower than that for the PEG spacer, indicating that the photograft spacers provide a higher accuracy of force determination.     

Molecular dynamics force probe simulations of antibody/antigen unbinding: entropic control and nonadditivity of unbinding forces

Unbinding of a spin-labeled dinitrophenyl (DNP) hapten from the monoclonal antibody AN02 F(ab) fragment has been studied by force probe molecular dynamics (FPMD) simulations. In our nanosecond simulations, unbinding was enforced by pulling the hapten molecule out of the binding pocket. Detailed inspection of the FPMD trajectories revealed a large heterogeneity of enforced unbinding pathways and a correspondingly large flexibility of the binding pocket region, which exhibited induced fit motions. Principal component analyses were used to estimate the resulting entropic contribution of approximately 6 kcal/mol to the AN02/DNP-hapten bond. This large contribution may explain the surprisingly large effect on binding kinetics found for mutation sites that are not directly involved in binding. We propose that such "entropic control" optimizes the binding kinetics of antibodies. Additional FPMD simulations of two point mutants in the light chain, Y33F and I96K, provided further support for a large flexibility of the binding pocket. Unbinding forces were found to be unchanged for these two mutants. Structural analysis of the FPMD simulations suggests that, in contrast to free energies of unbinding, the effect of mutations on unbinding forces is generally nonadditive.     

Energy landscape of streptavidin-biotin complexes measured by atomic force microscopy

The dissociation of ligand and receptor involves multiple transitions between intermediate states formed during the unbinding process. In this paper, we explored the energy landscape of the streptavidin-biotin interaction by using the atomic force microscope (AFM) to measure the unbinding dynamics of individual ligand-receptor complexes. The rupture force of the streptavidin-biotin bond increased more than 2-fold over a range of loading rates between 100 and 5000 pN/s. Moreover, the force measurements showed two regimes of loading in the streptavidin-biotin force spectrum, revealing the presence of two activation barriers in the unbinding process. Parallel experiments carried out with a streptavidin mutant (W120F) were used to investigate the molecular determinants of the activation barriers. From these experiments, we attributed the outer activation barrier in the energy landscape to the molecular interaction of the '3-4' loop of streptavidin that closes behind biotin.     

Immunoprecipitation distinguishes non-overlapping groups of snRNPs in Schizosaccharomyces pombe.

The large number of snRNAs in the fission yeast Schizosaccharomyces pombe can be divided into four non-overlapping groups by immunoprecipitation with antibodies directed against mammalian snRNP proteins. 1) Of the abundant snRNAs, anti-Sm sera precipitate only the spliceosomal snRNAs U1, U2, U4, U5 and U6. Surprisingly, three Sm-sera tested distinguish between U2, U4 and U5 and U1 from S.pombe; one precipitating only U1 and two precipitating U2, U4 and U5 but not U1. 2) A group of 11 moderately abundant snRNAs are not detectably precipitated by human anti-Sm sera, but are specifically precipitated by monoclonal antibody H57 specific for the human B/B' polypeptides. From Aspergillus nidulans this antibody also precipitates at least 12 snRNAs. 3) Anti-(U3)RNP sera do not precipitate the above snRNAs, but precipitate at least 6 further snRNAs, including the homologues of U3. Both the anti-(U3)RNP sera and H57 also efficiently precipitate a number of discrete non-capped RNAs. 4) A small number of additional snRNAs are not detectably precipitated by any anti-serum tested to date, further analysis may identify antisera specific for these snRNPs. Western blots of purified snRNP proteins were used to identify the S.pombe proteins responsible for these immunoprecipitations. Several Sm-sera decorate a 16.3kD protein which may be a D protein homologue, monoclonal H57 decorates a further protein of 16kD and an anti-(U3)RNP serum decorates the homologue of the 36kD U3-specific protein, fibrillarin.     

Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore

Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores.     

Uranyl photoprobing of a four-way DNA junction: evidence for specific metal ion binding.

Metal ions are very important in mediating the folding of nucleic acids, as exemplified by the folding of the four-way DNA junction into the stacked X-conformation. Uranyl ion-mediated photocleavage provides a method for the localization of high-affinity ion binding sites in nucleic acids, and we have applied this to the four-way DNA junction. We have made the following observations. (i) Uranyl ions (UO2(2+)) suppressed the reactivity of junction thymine bases against attack by osmium tetroxide, indicating that the uranyl ion induces folding of the junction into a stacked conformation. (ii) DNA located immediately at the point of strand exchange on the two exchanging strands was hypersensitive to uranyl photocleavage. The relative hypersensitivity was considerably accentuated when the photocleavage was carried out in the presence of citrate ions. This suggests the presence of a tight binding site for the uranyl ion in the junction. (iii) The same positions were significantly protected from uranyl cleavage by the presence of hexamminecobalt (III) or spermidine. These ions are known to induce the folded conformation of the four-way junction with high efficiency, suggesting a direct competition between the ions. By contrast, magnesium ions failed to generate a similar protection against photocleavage. These results suggest that the uranyl, hexamminecobalt (III) and spermidine ions compete for the same high affinity binding site on the junction. This site is located at the centre of the junction, at the point where the exchanging strands pass between the stacked helices. We believe that we have observed the first known example of a metal ion 'footprint' on a folded nucleic acid structure.