A rapid assay for affinity and kinetics of molecular interactions with nucleic acids

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) allows detection of protein interactions with low-molecular weight ligands based on separation of the protein-ligand complex by differential capillary action. Here, we present an application of DRaCALA to the study of nucleic acid-protein interactions using the Escherichia coli cyclic AMP receptor protein (CRP). CRP bound in DRaCALA specifically to (32)P-labeled oligonucleotides containing the consensus CRP binding site, but not to oligonucleotides with point mutations known to abrogate binding. Affinity and kinetic studies using DRaCALA yielded a dissociation constant and dissociation rate similar to previously reported values. Because DRaCALA is not subject to ligand size restrictions, whole plasmids with a single CRP-binding site were used as probes, yielding similar results. DNA can also function as an easily labeled carrier molecule for a conjugated ligand. Sequestration of biotinylated nucleic acids by streptavidin allowed nucleic acids to take the place of the protein as the immobile binding partner. Therefore, any molecular interactions involving nucleic acids can be tested. We demonstrate this principle utilizing a bacterial riboswitch that binds cyclic-di-guanosine monophosphate. DRaCALA is a flexible and complementary approach to other biochemical methods for rapid and accurate measurements of affinity and kinetics at near-equilibrium conditions.     

A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding. Application to Escherichia coli single-strand binding protein-nucleic acid interactions

We describe a general method for the analysis of ligand-macromolecule binding equilibria for cases in which the interaction is monitored by a change in a signal originating from the ligand. This method allows the absolute determination of the average degree of ligand binding per macromolecule without any assumptions concerning the number of modes or states for ligand binding or the relationship between the fractional signal change and the fraction of bound ligand. Although this method is generally applicable to any type of signal, we discuss the details of the method as it applies to the analysis of binding data monitored by a change in fluorescence of a ligand upon binding to a nucleic acid. We apply the analysis to the equilibrium binding of Escherichia coli single-strand binding (SSB) protein to single-stranded nucleic acids, which is monitored by the quenching of the intrinsic tryptophan fluorescence of the SSB protein. With this method, one can quantitatively determine the relationship between the fractional signal change of the ligand and the fraction of bound ligand, LB/LT, and rigorously test whether the signal change is directly proportional to LB/LT. For E. coli SSB protein binding to single-stranded nucleic acids in its (SSB)65 binding mode [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Chrysogelos, S., & Griffith, J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5803], we show that the fractional quenching of the SSB fluorescence is equal to the fraction of bound SSB.     

The DNA and RNA specificity of eilatin Ru(II) complexes as compared to eilatin and ethidium bromide

Eilatin-containing ruthenium complexes bind to a broad range of different nucleic acids including: calf thymus (CT) DNA, tRNA(Phe), polymeric RNAs and DNAs, and viral RNAs including the HIV-1 RRE and TAR. The nucleic acid specificity of Lambda- and Delta-[Ru(bpy)2eilatin]2+ have been compared to that of the 'free' eilatin ligand, and to the classic intercalating agent ethidium bromide. Interestingly, all four compounds appear to bind to nucleic acids by intercalation, but the trends in nucleic acid binding specificity are highly diverse. Unlike ethidium bromide, both eilatin and the eilatin-containing coordination complexes bind to certain single-stranded RNAs with high affinity (K(d) < or = 1 microM). Eilatin itself is selective for electron-poor polymeric purines, while the eilatin-coordination complexes exhibit preference for the polypyrimidine r(U). These results show how the binding specificity of an intercalating ligand can change upon its incorporation into an octahedral metal complex.     

Kinetics of nonspecific binding reactions of proteins with DNA flexible coils: site-based and molecule-based association reactions

Domain effects on the pseudo-first-order kinetics of the reversible and irreversible association of proteins or other ligands with nucleic acids containing multiple binding sites are treated using the classical reaction-diffusion equation applied to a spherical cell model of the nucleic acid solution and a diffuse-sphere model for the nucleic acid chain molecule. Both uniform and Gaussian distributions of chain segments are analyzed. In general, the details of the segment distribution do not have a major effect on the kinetics of association. Domain effects are best examined experimentally by determining the effect of the molecular weight of the nucleic acid on the kinetics of the association reaction. A theoretical framework is presented that permits such data to be analyzed simply. Kinetic studies over a wide range of nucleic acid molecular weights are required in order to separate the contributions of diffusion and reaction to the observed kinetics, and to determine the contributions of site-based and molecule-based elements to the rate constants.     

Nucleic acids exert a sequence-independent cooperative effect on sequence-dependent activation of Toll-like receptor 9

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. Although cell stimulation experiments demonstrate the preferential activation of TLR9 by CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions with respect to the ability of this receptor to bind nucleic acids in a sequence-specific manner. To address this apparent discrepancy, we report the purification of the soluble ectodomain of human TLR9 with characterization of its ligand binding properties. We observe that TLR9 has a high degree of specificity in its ability to bind nucleic acids that contain CpG dinucleotides as well as higher order motifs that mediate species-specific activation. However, TLR9 is also functionally influenced by nucleic acids in a sequence-independent fashion as both stimulatory and nonstimulatory nucleic acids sensitize TLR9 for in vitro ligand binding as well as in vivo activation. We propose a model in which receptor activation is achieved in a sequence-dependent manner, and sensitivity is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. This model bears resemblance to that recently proposed for Toll in that activation is a two-step process in which formation of a ligand-bound monomer precedes formation of the activated dimer. In each model receptor sensitivity is determined within the second step with the crucial distinction that Toll undergoes negative cooperativity, whereas TLR9 is sensitized through a positive cooperative effect.     

A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. I. Stability of the mercury-sulfhydryl bond and influence of the ligand structure on immunochemical detection of the hapten

The mechanisms underlying a new hybridocytochemical method, which is based on mercurated nucleic acid probes and their binding to sulfhydryl-hapten ligands, have been studied. Furthermore we developed a simple procedure for the preparation of mercurated probes at a microgram scale. Nucleic acids immobilized on Sephadex beads have been immunochemically detected after hybridization with mercurated probes and binding of the sulfhydryl-hapten ligand trinitrophenyl-glutathione. In this system, the method proved to be specific and sensitive. However, the same procedure, when applied in situ, failed to give a positive result. ELISA experiments showed that these results cannot be attributed to a suboptimal immunochemical detection of the ligand. Chromatographic analysis of mercurated polynucleotide-ligand complexes revealed, however, an unexpected lability of the mercury-sulfhydryl bond. Under non-equilibrium conditions, as present during a cytochemical washing procedure, the mercury-sulfhydryl b ond was found to dissociate rapidly. On basis of these results the hypothesis was forwarded that the bond between mercurated nucleic acids immobilized on Sephadex and the ligand was stabilized by the positive charge of the Sephadex matrix. This charge was introduced during the cyanogen bromide activation and inactivation necessary for the covalent coupling of nucleic acids to Sephadex. In situ, however, no such positive charges are present. By reversing the charge of the ligand we expected to stabilize the mercury-sulfhydryl bond. In a subsequent paper data are presented that confirm this hypothesis.     

Insulin-like growth factor-I signaling in smooth muscle cells is regulated by ligand binding to the 177CYDMKTTC184 sequence of the beta3-subunit of alphaVbeta3

The response of smooth muscle cells to IGF-I requires ligand occupancy of the alphaVbeta3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-alphaVbeta3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177-184 ((177)CYDMKTTC(184)) within the extracellular domain of beta3 have been proposed to confer the ligand specificity of alphaVbeta3; therefore, we hypothesized that ligand binding to the 177-184 cysteine loop of beta3 may be an important regulator of the cross talk between alphaVbeta3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the beta3 subunit of alphaVbeta3 (i.e. amino acids 177-184) blocked Vn binding to the beta3 subunit of alphaVbeta3 and correspondingly beta3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of beta3 in which three critical residues within the 177-184 sequence were altered beta3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177-184 sequence of beta3 is necessary for Vn binding to alphaVbeta3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I.     

In vitro preparation of amelogenin nanoparticles carrying nucleic acids

Amelogenin, a matrix protein involved in biomineralization of enamel, can self-assemble to form nanospheres in a pH-dependent manner. Nucleic acids (single-stranded, double-stranded, and plasmid DNA, as well as RNA) could be co-precipitated with amelogenin, demonstrating a strong binding of nucleic acids to amelogenin. The amounts of co-precipitated nucleic acids were analyzed and binding levels upto 90 μg DNA/mg amelogenin was achieved. The co-precipitation could also be carried out in a bacterial cell homogenate, and no bacterial proteins were found in the amelogenin aggregates, suggesting specificity for nucleic acid binding. Dynamic light scattering showed that amelogenin nanosphere structure is maintained upon DNA binding with an upto 2.6 nm increase in diameter. The reported binding of nucleic acids to amelogenin can be explored practically for nucleic acid separation.     

Theoretical modeling of DNA-monocationic lexitropsin complexation: influence of ligand binding on DNA curvature

A theoretical study is presented on the complexation to DNA of a monocationic lexitropsin. Energetics and the structures of the complexes formed are analyzed for three base pair sequences of a nucleic acid octamer. The influence of the ligand binding on the nucleic acid conformation is analysed in detail. It is found that whereas the uncomplexed nucleic acid segments have very irregular structures with an overall curvature varying between 15 degrees and 20 degrees, the DNA structure becomes more regular and the curvature is strongly reduced upon the binding of a monocationic lexitropsin.     

Competition dialysis: a method for the study of structural selective nucleic acid binding

Competition dialysis is a powerful new tool for the discovery of ligands that bind to nucleic acids with structural- or sequence-selectivity. The method is based on firm thermodynamic principles and is simple to implement. In the competition dialysis experiment, an array of nucleic acid structures and sequences is dialyzed against a common test ligand solution. After equilibration, the amount of ligand bound to each structure or sequence is determined by absorbance or fluorescence measurements. Since all structures and sequences are in equilibrium with the same free ligand concentration, the amount bound is directly proportional to the ligand binding affinity. Competition dialysis thus provides a direct and quantitative measure of selectivity, and unambiguously identifies which of the samples within the array are preferred by a particular ligand. We describe here the third generation implementation of the method, in which competition dialysis was adapted for use in a 96-well plate format. In this format, we have been able to greatly expand the array of nucleic acid structures studied, and now can routinely study the interactions of a ligand of interest with 46 different structures and sequences.     

Sequence- and structural-selective nucleic acid binding revealed by the melting of mixtures

A simple method for the detection of sequence- and structural-selective ligand binding to nucleic acids is described. The method is based on the commonly used thermal denaturation method in which ligand binding is registered as an elevation in the nucleic acid melting temperature (T_m). The method can be extended to yield a new, higher -throughput, assay by the simple expediency of melting designed mixtures of polynucleotides (or oligonucleotides) with different sequences or structures of interest. Upon addition of ligand to such mixtures at low molar ratios, the T_m is shifted only for the nucleic acid containing the preferred sequence or structure. Proof of principle of the assay is provided using first a mixture of polynucleotides with different sequences and, second, with a mixture containing DNA, RNA and two types of DNA:RNA hybrid structures. Netropsin, ethidium, daunorubicin and actinomycin, ligands with known sequence preferences, were used to illustrate the method. The applicability of the approach to oligonucleotide systems is illustrated by the use of simple ternary and binary mixtures of defined sequence deoxyoligonucleotides challenged by the bisanthracycline WP631. The simple mixtures described here provide proof of principle of the assay and pave the way for the development of more sophisticated mixtures for rapidly screening the selectivity of new nucleic acid binding compounds.     

The evolution of bat nucleic acid‐sensing Toll‐like receptors

We characterized the nucleic acid‐sensing Toll‐like receptors (TLR) of a New World bat species, the common vampire bat (Desmodus rotundus), and through a comparative molecular evolutionary approach searched for general adaptation patterns among the nucleic acid‐sensing TLRs of eight different bats species belonging to three families (Pteropodidae, Vespertilionidae and Phyllostomidae). We found that the bat TLRs are evolving slowly and mostly under purifying selection and that the divergence pattern of such receptors is overall congruent with the species tree, consistent with the evolution of many other mammalian nuclear genes. However, the chiropteran TLRs exhibited unique mutations fixed in ligand‐binding sites, some of which involved nonconservative amino acid changes and/or targets of positive selection. Such changes could potentially modify protein function and ligand‐binding properties, as some changes were predicted to alter nucleic acid binding motifs in TLR 9. Moreover, evidence for episodic diversifying selection acting specifically upon the bat lineage and sublineages was detected. Thus, the long‐term adaptation of chiropterans to a wide variety of environments and ecological niches with different pathogen profiles is likely to have shaped the evolution of the bat TLRs in an order‐specific manner. The observed evolutionary patterns provide evidence for potential functional differences between bat and other mammalian TLRs in terms of resistance to specific pathogens or recognition of nucleic acids in general.     

Nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen

The N terminal region of hepatitis delta antigen (HDAg), referred to here as NdAg, has a nucleic acid chaperone activity that modulates the ribozyme activity of hepatitis delta virus (HDV) RNA and stimulates hammerhead ribozyme catalysis. We characterized the nucleic acid binding properties of NdAg, identified the structural and sequence domains important for nucleic acid binding, and studied the correlation between the nucleic acid binding ability and the nucleic acid chaperone activity. NdAg does not recognize the catalytic core of HDV ribozyme specifically. Instead, NdAg interacts with a variety of nucleic acids and has higher affinities to longer nucleic acids. The studies with RNA homopolymers reveal that the binding site size of NdAg is around nine nucleotides long. The extreme N terminal portion of NdAg, the following coiled-coil domain and the basic amino acid clusters in these regions are important for nucleic acid binding. The nucleic acid–NdAg complex is stabilized largely by electrostatic interactions. The formation of RNA–protein complex appears to be a prerequisite for facilitating hammerhead ribozyme catalysis of NdAg and its derivatives. Mutations that reduce the RNA binding activity or high ionic strength that destabilizes the RNA–protein complex, reduce the nucleic acid chaperone activity of NdAg.     

Retroviral nucleocapsid proteins display nonequivalent levels of nucleic acid chaperone activity

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a nucleic acid chaperone that facilitates the remodeling of nucleic acids during various steps of the viral life cycle. Two main features of NC's chaperone activity are its abilities to aggregate and to destabilize nucleic acids. These functions are associated with NC's highly basic character and with its zinc finger domains, respectively. While the chaperone activity of HIV-1 NC has been extensively studied, less is known about the chaperone activities of other retroviral NCs. In this work, complementary experimental approaches were used to characterize and compare the chaperone activities of NC proteins from four different retroviruses: HIV-1, Moloney murine leukemia virus (MLV), Rous sarcoma virus (RSV), and human T-cell lymphotropic virus type 1 (HTLV-1). The different NCs exhibited significant differences in their overall chaperone activities, as demonstrated by gel shift annealing assays, decreasing in the order HIV-1 ~ RSV > MLV HTLV-1. In addition, whereas HIV-1, RSV, and MLV NCs are effective aggregating agents, HTLV-1 NC, which exhibits poor overall chaperone activity, is unable to aggregate nucleic acids. Measurements of equilibrium binding to single- and double-stranded oligonucleotides suggested that all four NC proteins have moderate duplex destabilization capabilities. Single-molecule DNA-stretching studies revealed striking differences in the kinetics of nucleic acid dissociation between the NC proteins, showing excellent correlation between nucleic acid dissociation kinetics and overall chaperone activity.