Nucleic binding affinity of bacteriophage T4 gene 32 protein in the cooperative binding mode

This study reports on various parameters which affect the binding stoichiometry for complexes of bacteriophage T4 gene 32 protein (P32) and single stranded polynucleotides (determined by UV absorbance and fluorescence quenching) and presents results of a quantitative electron spin resonance assay to determine physiologically effective binding affinity differences of nucleic acid binding proteins. The assay employs macromolecular spin probes (spin-labeled nucleic acids) which are used to determine the fraction of saturation in competition experiments with unlabeled nucleic acids. It was found that the fraction of complexed spin-labeled polynucleotides can be directly monitored by ESR with a two-component analysis approach when ligands such as poly(L-lysine), gene 5 protein (P5) of filamentous bacteriophage fd, and gene 32 protein (P32) of bacteriophage T4 are used. The ESR data unequivocally show that: 1) the binding stoichiometry for poly(L-lysine), P5 and P32 is nucleotide/lysine, 4 nucleotides/P5 monomer, and 10 nucleotides/P32 monomer, respectively; and 2) under physiologically relevant buffer conditions the relative affinity of P32 in the cooperative binding mode for polythymidylic acid is about 4 times greater than for polydeoxyinosinic acid and about 12 times greater than for polyinosinic acid, and the relative affinity of P32 for polydeoxyinosinic acid is about 3 times greater than for polyinosinic acid.     

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids: I. Characterization of the binding interactions

In this paper we examine molecular details of the interaction of bacteriophage T4-coded gene 32 protein with oligo- and polynucleotides. It is shown that the binding affinity (K_oligo) of oligonucleotides of length (l) from two to eight nucleotide residues for gene 32 protein is essentially independent of base composition or sugar type. This binding also shows little dependence on salt concentration and on oligonucleotide length; even the expected statistical length factor in K_oligo is not observed, suggesting that binding occurs at the end of the oligonucleotide lattice and that the oligonucleotide is not free to move across the binding site. Co-operative (contiguous) or isolated binding of gene 32 protein to polynucleotides is very different; here binding is highly salt dependent ($\text{? log Kω}\text{? log}\text{[NaCl]}\text{～- ?7}\text{)}$ and essentially stoichiometric at salt concentrations less than ~0.2 m (for poly(rA)). Binding becomes much weaker and the binding isotherms appear typically co-operative (sigmoid) in protein concentration at higher salt concentrations. We demonstrate, by fitting the co-operative binding isotherms to theoretical plots at various salt concentrations and also by measuring binding at very low protein binding density (ν), that the entire salt dependence of Kω is in the intrinsic binding constant (K); the co-operativity parameter (ω) is essentially independent of salt concentration. Furthermore, by determining titration curves in the presence of salts containing a series of different anions and cations, it is shown that the major part of the salt dependence of the gene 32 protein-polynucleotide interaction is due to anion (rather than to cation) displacement effects. Binding parameters of oligonucleotides of length sufficient to bind two or more gene 32 protein monomers show behavior intermediate between the oligonucleotide and the polynucleotide binding modes. These different binding modes probably reflect different conformations of the protein; the results are analyzed to produce a preliminary molecular model of the interactions of gene 32 protein with nucleic acids in its different binding modes.     

Recognition of chemically damaged DNA by the gene 32 protein from bacteriophage T4

We have used fluorescence spectroscopy to investigate the binding of gene 32 protein from bacteriophage T4 to DNA which has been chemically modified with carcinogens or antitumor drugs. This protein exhibits a high specificity for single-stranded nucleic acids and binds more efficiently to DNA modified either with cis-diaminodichloroplatinum(II) or with aminofluorene derivatives than to native DNA. This increased affinity is related to the formation of locally unpaired regions which are strong binding sites for the single-strand binding protein. In contrast, gene 32 protein has the same affinity for native DNA, DNA containing methylated purines and DNA that has reacted with trans-diaminodichloroplatinum(II) or with chlorodiethylenetriaminoplatinum(II) chloride. These types of damage do not induce a sufficient structural change to allow gene 32 protein binding. Depurination of DNA does not create binding sites for the T4 gene 32 protein but nicked apurinic sites are strong ligands for the protein. This T4 single-strand binding protein does not exhibit a significantly increased affinity for nicked DNA as compared with native DNA. These results are discussed with respect to the recognition of DNA damage by proteins involved in DNA repair and to the possible role of single-strand binding proteins in DNA repair mechanisms.     

Theory of electrostatically regulated binding of T4 gene 32 protein to single- and double-stranded DNA

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA binding protein, which is essential for DNA replication, recombination, and repair. In a recent article, we described a new method using single DNA molecule stretching measurements to determine the noncooperative association constants K(ds) to double-stranded DNA for gp32 and *I, a truncated form of gp32. In addition, we developed a single molecule method for measuring K(ss), the association constant of these proteins to single-stranded DNA. We found that in low salt both K(ds) and K(ss) have a very weak salt dependence for gp32, whereas for *I the salt dependence remains strong. In this article we propose a model that explains the salt dependence of gp32 and *I binding to single-stranded nucleic acids. The main feature of this model is the strongly salt-dependent removal of the C-terminal domain of gp32 from its nucleic acid binding site that is in pre-equilibrium to protein binding to both double-stranded and single-stranded nucleic acid. We hypothesize that unbinding of the C-terminal domain is associated with counterion condensation of sodium ions onto this part of gp32, which compensates for sodium ion release from the nucleic acid upon its binding to the protein. This results in the salt-independence of gp32 binding to DNA in low salt. The predictions of our model quantitatively describe the large body of thermodynamic and kinetic data from bulk and single molecule experiments on gp32 and *I binding to single-stranded nucleic acids.     

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids: II. Specificity of binding to DNA and RNA

In this paper we examine the specificity of the co-operative binding (in the polynucleotide mode) of bacteriophage T4-coded gene 32 protein to synthetic and natural single-stranded nucleic acids differing in base composition and sugar type. It is shown by competition experiments in a tight-binding (low salt) environment that there is a high degree of binding specificity under these (protein-limiting) conditions, with one type of nucleic acid lattice binding gene 32 protein to saturation before any binding to the competing lattice takes place; it is also shown that the same differential specificities apply at high salt concentrations. Procedures developed in the preceding paper (Kowalczykowski et al., 1980) are used to measure the net binding affinities (Kω) of gene 32 protein to a variety of polynucleotides, as well as to determine individual values of K and ω for some systems. For all polynucleotides, virtually the entire specificity and salt dependence of binding of Kω appears to be in K. In ~0.2 m-NaCl, the net binding affinities (Kω) range from ~10⁶ to ~10¹¹m^?1; in order of increasing affinities we find: poly(rC) < poly(rU) < poly(rA) < poly(dA) < poly(dC) < poly(dU) < poly(rI) < poly(dI) < poly-(dT). In general, Kω for a particular homopolyribonucleotide at constant salt concentration is 10¹ to 10⁴smaller than Kω for the corresponding homopoly-deoxyribopolynucleotide. Values of Kω for randomly copolymerized polynucleotides and for natural DNA fall at the compositionally weighted average of the values for the individual homopolynucleotides (except for poly(dT), which appears to bind somewhat tighter), indicating that the net affinity represents the sum of the binding free energy contributions of the individual nucleotides. It is shown that these results, on a competition basis under physiological salt conditions, can account quantitatively for the autogenous regulation of the synthesis of gene 32 protein at the translational level (Russel et al., 1976; Lemaire et al., 1978). In addition, these results suggest possible mechanisms by which gene 32 messenger RNA might be specifically recognized (by gene 32 protein) and functionally discriminated from the other mRNAs of phage T4.     

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids: III. Binding properties of two specific proteolytic digestion products of the protein (G32P1I and G32P1III)

Brief treatment of gene 32 protein with proteolytic enzymes produces two specific digestion products in good yield (Moise & Hosoda, 1976). One, representing the native protein with ~60 amino acid residues removed from the C-terminus, is G32P¹I. The other, for which ~20 amino acid residues have been removed from the N-terminus in addition to the 60 residues from the C-terminus, is G32P¹III. Both of these specific “core” fragments of gene 32 protein have been isolated and purified, and their binding properties to single-stranded oligo- and polynucleotides have been studied. We find that the binding properties of G32P¹I are relatively little changed from those characteristic of the native gene 32 protein: (1) the apparent binding constants to short (l = 2 to 8) oligonucleotides are independent of lattice length and essentially independent of base and sugar composition, but do show an increased salt dependence of binding relative to that of the native protein; (2) the intrinsic association constants (K) for polynucleotides binding in the co-operative mode show the same binding specificities as seen with the native protein, but with absolute values increased two to fourfold; (3) the polynucleotide binding co-operativity parameter (ω^?2 × 10³) and the binding site size (n ～-7 nucleotide residues) are the same as for the native protein; (4) essentially the entire salt dependence of the net affinity (Kω) remains in K. However, unlike native gene 32 protein, G32P¹I can melt native DNA to equilibrium (Hosoda et al., 1974; Greve et al., 1978); this suggests that the kinetic pathways for DNA melting by these two species must differ, since the changes in equilibrium binding parameters measured here are far too small to account for the differences in melting behavior. In contrast to G32P¹I, for G32P¹III we find that: (1) binding is non-cooperative (ω ～-1); (2) the binding site size (n) for the protein has decreased by one to two nucleotide residues relative to that characteristic of the native protein and G32P¹I; (3) binding to short (l = 2 to 8) oligonucleotides is length and salt concentration dependent; (4) while binding to polynucleotides continues to show approximately the same base composition dependence as the native protein, the absolute values of K are somewhat different and the salt concentration dependencies of K are less. Polynucleotide ultraviolet light and circular dichroism spectra obtained in the presence of G32P¹I and G32P¹III are indistinguishable from those measured with the native protein at similar binding densities, indicating that all three protein species distort the polynucleotide lattice to comparable extents.These results are combined with the equilibrium binding data for native gene 32 protein (Kowalczykowski et al., 1980a: Newport et al., 1980) to obtain further insight into the molecular details of the interactions of this protein with its nucleic acid binding substrates.     

An EPR study to determine the relative nucleic acid binding affinity of single-stranded DNA-binding protein from Escherichia coli.

A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported. Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer. Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl). The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM. It is, therefore, metastable and forms only under reverse titration conditions in low NaCl. The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex. The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V. and Ireland, J.C. (1982) J. Biol. Chem. 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C. and Bobst, E.V. (1984) J. Biol. Chem. 259, 2130) by EPR competition assays. In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein. The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n.     

YbiB from Escherichia coli,the Defining Member of the Novel TrpD2 Family of Prokaryotic DNA-binding Proteins

We present the crystal structure and biochemical characterization of Escherichia coli YbiB, a member of the hitherto uncharacterized TrpD2 protein family. Our results demonstrate that the functional diversity of proteins with a common fold can be far greater than predictable by computational annotation. The TrpD2 proteins show high structural homology to anthranilate phosphoribosyltransferase (TrpD) and nucleoside phosphorylase class II enzymes but bind with high affinity (K_D = 10–100 nm) to nucleic acids without detectable sequence specificity. The difference in affinity between single- and double-stranded DNA is minor. Results suggest that multiple YbiB molecules bind to one longer DNA molecule in a cooperative manner. The YbiB protein is a homodimer that, therefore, has two electropositive DNA binding grooves. But due to negative cooperativity within the dimer, only one groove binds DNA in in vitro experiments. A monomerized variant remains able to bind DNA with similar affinity, but the negative cooperative effect is eliminated. The ybiB gene forms an operon with the DNA helicase gene dinG and is under LexA control, being induced by DNA-damaging agents. Thus, speculatively, the TrpD2 proteins may be part of the LexA-controlled SOS response in bacteria.     

Specificity of translational regulation by two DNA-binding proteins

The gene V protein of the filamentous bacteriophages f1, fd and M13, and the gene 32 protein of bacteriophage T4 share the property of binding strongly and co-operatively to single-stranded nucleic acids, especially DNA. Moreover, both are capable of repressing the translation of specific mRNAs (gene 32 protein its own, and gene V protein that of the filamentous phage gene II), both in vivo and in vitro. If the mechanism of repression by either of these proteins were based solely on its ability to bind single strands co-operatively, then the other would be expected to mimic or interfere with its effect in vitro. We have found no such mimicry or interference, even at protein concentrations high enough to have substantial non-specific effects on translation. This suggests that the sites of repression on the mRNAs must offer something other than simple “unstructuredness” for binding and repression to occur.     

Nucleic acid-binding specificity of human FUS protein

FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with K_d^app values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners.     

Recognition of specific and nonspecific DNA by human lactoferrin

The general principles of recognition of nucleic acids by proteins are among the most exciting problems of molecular biology. Human lactoferrin (LF) is a remarkable protein possessing many independent biological functions, including interaction with DNA. In human milk, LF is a major DNase featuring two DNA‐binding sites with different affinities for DNA. The mechanism of DNA recognition by LF was studied here for the first time. Electrophoretic mobility shift assay and fluorescence measurements were used to probe for interactions of the high‐affinity DNA‐binding site of LF with a series of model‐specific and nonspecific DNA ligands, and the structural determinants of DNA recognition by LF were characterized quantitatively. The minimal ligands for this binding site were orthophosphate (K_i = 5 mM), deoxyribose 5'‐phosphate (K_i = 3 mM), and different dNMPs (K_i = 0.56–1.6 mM). LF interacted additionally with 9–12 nucleotides or nucleotide pairs of single‐ and double‐stranded ribo‐ and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleoside phosphate groups. Such nonspecific interactions of LF with noncognate single‐ and double‐stranded d(pN)₁₀ provided ~6 to ~7.5 orders of magnitude of the enzyme affinity for any DNA. This corresponds to the Gibbs free energy of binding (ΔG⁰) of ?8.5 to ?10.0 kcal/mol. Formation of specific contacts between the LF and its cognate DNA results in an increase of the DNA affinity for the enzyme by approximately 1 order of magnitude (K_d = 10 nM; ΔG⁰ ≈ ?11.1 kcal/mol). A general function for the LF affinity for nonspecific d(pN)_n of different sequences and lengths was obtained, giving the K_d values comparable with the experimentally measured ones. A thermodynamic model was constructed to describe the interactions of LF with DNA. Copyright © 2013 John Wiley & Sons, Ltd.     

Cold shock domain protein from Philosamia ricini prefers single-stranded nucleic acids binding

The cold shock proteins are evolutionarily conserved nucleic acid-binding proteins. Their eukaryotic homologs are present as cold shock domain (CSD) in Y-box proteins. CSDs too share striking similarity among different organisms and show nucleic acid binding properties. The purpose of the study was to investigate the preferential binding affinity of CSD protein for nucleic acids in Philosamia ricini. We have cloned and sequenced the first cDNA coding for Y-box protein in P. ricini; the sequence has been deposited in GenBank. Comparative genomics and phylogenetic analytics further confirmed that the deduced amino acid sequence belongs to the CSD protein family. A comparative study employing molecular docking was performed with P. ricini CSD, human CSD, and bacterial cold shock protein with a range of nucleic acid entities. The results indicate that CSD per se exhibits preferential binding affinity for single-stranded RNA and DNA. Possibly, the flanking N- and C-terminal domains are additionally involved in interactions with dsDNA or in conferring extra stability to CSD for improved binding.     

Corticosteroid modulation of muscarinic receptors in rat myocardial membranes

Rat ventricular myocardial membanes contain muscarinic acetylcholine receptors which can be identified by binding of the muscarinic antagonist (-)-[³H]quinuclidinyl benzilate. Scatchard analysis of saturation binding data revealed binding to a single class of non-cooperative sites (0.693 pmol/mg protein) with high affinity (i.e. with an equilibrium dissociation constant of 0.24 nM). Competition binding curves of the agonist carbamylholine were shallow (with a Hill coefficient, n_H of 0.71) for membranes of untreated rats, suggesting the presence of two receptor subpopulations with different agonist affinity. These curves were steeper (n_H = 0.86) for adrenalectomized animals and more shallow (n_H = 0.62) for hydrocortisone-treated animals. In contrast, both treatments did not affect the total receptor number. This suggests that corticosteroids are required for the myocardial muscarinic receptors to adopt high agonist affinity. However, the inhibition of adenylate cyclase by muscarinic agonists disappeared after both corticosteroid treatment and adrenalectomy. But agonist receptor binding could still be modulated by guanine nucleotides. This indicates that both high and low affinity froms of muscarinic receptors induced by altered corticosteroid states retain functional coupling with the inhibitory nucleotide binding site, but are uncoupled from the adenylate cyclase catalytic subunit, C.     

The (gt)n(ga)m containing intron 2 of HLA-DRB alleles binds a zinc-dependent protein and forms non B-DNA structures

We studied protein binding and structural features of perfect and imperfect composite (gt)_n(ga)_m blocks from different HLA-DRB1 alleles in their original genomic and artificial environments. The major retarded protein/DNA complex of the genomic (gt)_n(ga)_m fragments comprises a zinc-dependent protein present in nuclear extracts from different cell types. The protein binding is characterized by moderate affinities independent of the polymorphic form of the physiological microsatellite allele. The binding affinity depends on the 5′ and 3′ adjacent single copy parts. DNase I footprinting of genome-derived fragments revealed that the 5′ adjacent sequence and the (gt)_n repeat are preferentially protected on the (gt)_n(ga)_m strand. Comparing three alleles, a regular pattern of footprints was not detectable in the (gt)_n part, indicating that the zinc-dependent protein recognizes structural rather than sequence-specific features in this region. Chemical probing resulted in a pattern characteristic for Z-DNA in the (gt)_n tract of the fragments. However, EMSA experiments using the Z-DNA specific monoclonal antibody mABZ-22 did not prove the presence of Z-DNA. As demonstrated by chemical modifications of the different (ga)_m targets, only one of three (gt)_n(ga)_m fragments formed intramolecular triplexes of the type H-y3 and H-y5. DNase I footprinting revealed only weak protection, if any, in the homopurine tract. Rather, the (tc)_m strands are hypersensitive for DNase I. This is probably due to structural conversions into intramolecular *H-triplexes after binding of HIZP.     

Purification and characterization of folate binding proteins from rat placenta

Rat placenta contains virtually no unsaturated (i.e., apo-form) folate binding protein. However, by lowering the pH of a solubilized membrane preparation of this tissue to 3.5, the endogenous bound folate was dissociated from the protein and adsorbed to charcoal. The apo-form of the folate binding protein thus obtained was purified by affinity chromatography using pteroylglutamic acid covalently coupled to Sepharose 4B. A single protein band with an apparent M_r of 36 000 was observed by SDS-polyacrylamide gel electrophoresis of the eluate from the affinity matrix. Western blot of this preparation using a rabbit antiserum raised with the affinity eluate also identified a single 36 kDa protein band. However, peptide sequencing of the N-terminal region of the proteins in the affinity eluate established that it contained two homologous proteins. Computer alignment of the first 22 N-terminal amino acids of each rat placental protein with human, bovine milk and mouse folate binding proteins showed 50–64% identical homology and 27% homology when the eight proteins were aligned together. The affinity of both rat proteins is highest for pteroylglutamic acid (K_a = 1.6 − 109 l/mol) lower for N⁵-methyltetrahydrofolate and substantially lower for N⁵-formyltetrahydrofolate. In the dose-response range studied there was no apparent affinity for methotrexate. The folate binding proteins could be released from a preparation of placental membranes using phospholipase C indicating that these proteins belong to the class of proteins anchored to the plasma membrane by a glycosyl phosphatidylinositol adduct.     

Identification of a 14mer RNA that recognizes and binds flavin mononucleotide with high affinity

Aptamers are nucleic acids developed by in vitro evolution techniques that bind to specific ligands with high affinity and selectivity. Despite such high affinity and selectivity, however, in vitro evolution does not necessarily reveal the minimum structure of the nucleic acid required for selective ligand binding. Here, we show that a 35mer RNA aptamer for the cofactor flavin mononucleotide (FMN) identified by in vitro evolution can be computationally evolved to a mere 14mer structure containing the original binding pocket and eight scaffolding nucleotides while maintaining its ability to bind in vitro selectively to FMN. Using experimental and computational methodologies, we found that the 14mer binds with higher affinity to FMN (K_D ~ 4 µM) than to flavin adenine dinucleotide (K_D ~ 12 µM) or to riboflavin (K_D ~ 13 µM),despite the negative charge of FMN. Different hydrogen-bond strengths resulting from differing ring-system electron densities associated with the aliphatic-chain charges appear to contribute to the selectivity observed for the binding of the 14mer to FMN and riboflavin. Our results suggest that high affinity and selectivity in ligand binding is not restricted to large RNAs, but can also be a property of extraordinarily short RNAs.     

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.     

Structural Requirements for the Procoagulant Activity of Nucleic Acids

Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects.     

The Use of the Free Metal – Temperature ‘Phase Diagrams’ for Studies of Single Site Metal Binding Proteins

Typical physico-chemical studies of metal binding proteins are usually aimed at determination of the metal binding constant K for a native protein (K _n), while the significance of the K value for the thermally denatured protein (K _u) is usually underestimated. Meanwhile, metal binding induced shift of thermal denaturation transition of a single site metal binding protein is defined by K _n to K _u ratio, implying that knowledge of both K values is required for full characterization of the system. In the present work, the most universal approach to the studies of single site metal binding proteins, namely construction of a protein “phase diagram” in coordinates of free metal ion concentration – temperature, is considered in detail. The detailed algorithm of construction of the phase diagrams along with underlying mathematic procedures developed here may be of use for studies of other simple protein-target type systems, where target represents low molecular weight ligand. Analysis of the simplest protein-ligand system reveals that thermodynamic properties of apo-protein dictate the maximal possible increase of its affinity to any simple ligand upon thermal denaturation of the protein. Experimental and general problems coupled with the use of the phase diagrams are discussed.     

Crystal Structure of Interleukin-6 in Complex with a Modified Nucleic Acid Ligand

IL-6 is a secreted cytokine that functions through binding two cell surface receptors, IL-6Rα and gp130. Because of its involvement in the progression of several chronic inflammatory diseases, IL-6 is a target of pharmacologic interest. We have recently identified a novel class of ligands called SOMAmers (S low Off-rate Modified Aptamers) that bind IL-6 and inhibit its biologic activity. SOMAmers exploit the chemical diversity of protein-like side chains assembled on flexible nucleic acid scaffolds, resulting in an expanded repertoire of intra- and intermolecular interactions not achievable with conventional aptamers. Here, we report the co-crystal structure of a high affinity SOMAmer (K_d = 0.20 nm) modified at the 5-position of deoxyuridine in a complex with IL-6. The SOMAmer, comprised of a G-quartet domain and a stem-loop domain, engages IL-6 in a clamp-like manner over an extended surface exhibiting close shape complementarity with the protein. The interface is characterized by substantial hydrophobic interactions overlapping the binding surfaces of the IL-6Rα and gp130 receptors. The G-quartet domain retains considerable binding activity as a disconnected autonomous fragment (K_d = 270 nm). A single substitution from our diversely modified nucleotide library leads to a 37-fold enhancement in binding affinity of the G-quartet fragment (K_d = 7.4 nm). The ability to probe ligand surfaces in this manner is a powerful tool in the development of new therapeutic reagents with improved pharmacologic properties. The SOMAmer·IL-6 structure also expands our understanding of the diverse structural motifs achievable with modified nucleic acid libraries and elucidates the nature with which these unique ligands interact with their protein targets.