Synthesis and DNA binding properties of terminally modified peptide nucleic acids

PNAs with terminal modifications of varying structure and charge were synthesized and their binding to DNA was studied. A variation in thermal stability of 19. 8 degrees C has been observed between the least and the most stable PNA-DNA duplexes. The most stable duplex melts 7.7 degrees C higher than the duplex of the corresponding non-modified PNA and complementary DNA. It has been shown that sequence fidelity of the PNA conjugate having the highest DNA affinity is significantly better than that of non-modified PNA. The results obtained can be used for the design of PNA probes, whose binding to DNA is sequence independent.     

Circular dichroism determination of class I MHC-peptide equilibrium dissociation constants

Class I major histocompatibility complex (MHC) molecules bind peptides derived from degraded proteins for display to T cells of the immune system. Peptides bind to MHC proteins with varying affinities, depending upon their sequence and length. We demonstrate that the thermal stability of the MHC-peptide complex depends directly on peptide binding affinity. We use this correlation to develop a convenient method to determine peptide dissociation constants by measuring MHC-peptide complex stability using thermal denaturation profiles monitored by circular dichroism.     

Dissecting the oligonucleotide binding properties of a disordered chaperone protein using surface plasmon resonance

We have used surface plasmon resonance to investigate the nucleic acid binding properties of the core protein of hepatitis C virus, a disordered protein believed to chaperone the genomic RNA. It was previously shown that a peptide (peptide E) corresponding to the association of two basic clusters of core enhances the annealing and the dimerization of nucleic acid fragments derived from a stem loop (SL2) in the 3′ untranslated region of the hepatitis C virus genome. However, strong aggregation of nucleic acids by core or peptide E in the excess of the latter precluded the characterization of their binding parameters up to now. By careful design of surface plasmon resonance experiments, we obtained accurate binding parameters for the interaction of peptide E with SL2-derived oligonucleotides of different lengths and sequences, in form of stem-loop, duplex or strand. Peptide E was found to bind in a salt dependent manner to all oligonucleotides assayed. Affinity data identify at least two binding modes, of which one is independent of sequence/structure, and the other is specific to the SL2 stem-loop fold. Stoichiometry data support a multi-motif binding model allowing formation of higher-order complexes. We propose that the modular binding mode demonstrated for structured RNA-binding proteins also applies to this disordered chaperone and is relevant to its activity.     

Selective recognition of pyrimidine-pyrimidine DNA mismatches by distance-constrained macrocyclic bis-intercalators

Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas the acyclic model compounds are much less selective and strongly bind to the matched DNA. Moreover, the results from thermal denaturation experiments are in very good agreement with the binding affinities obtained by ESI-MS and FID measurements. The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine–pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch, as shown by competitive fluorescence titrations. Altogether, our results demonstrate that macrocyclic distance-constrained bis-intercalators are efficient and selective mismatch-binding ligands that can interfere with mismatch-binding enzymes.     

Investigation of some properties of oligodeoxynucleotides containing 4'-thio-2'-deoxynucleotides: duplex hybridization and nuclease sensitivity.

The thermal stabilities of the duplexes formed between 4'-thio-modified oligodeoxynucleotides and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligodeoxynucleotides. A 16mer oligodeoxynucleotide containing 10 contiguous 4'-thiothymidylate modifications formed a less stable duplex with the DNA target (deltaTm/modification -1.0 degrees C) than the corresponding unmodified oligodeoxynucleotide. However, when the same oligodeoxynucleotide was bound to the corresponding RNA target, a small increase in Tm was observed (deltaTm/modification +0.16 degrees C) when compared with the unmodified duplex. A study to identify the specificity of an oligodeoxynucleotide containing a 4'-thiothymidylate modification when forming a duplex with DNA or RNA containing a single mismatch opposite the modification found the resulting Tms to be almost identical to the wild-type duplexes, demonstrating that the 4'-thio-modification in oligodeoxynucleotides has no deleterious effect on specificity. The nuclease stability of 4'-thio-modified oligodeoxynucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. No significant resistance to degradation by the exonuclease SVPD was observed when compared with the corresponding unmodified oligodeoxynucleotide. However, 4'-thio-modified oligodeoxynucleotides were found to be highly resistant to degradation by the endonuclease S1. It was also demonstrated that 4'-thio-modified oligodeoxynucleotides elicit Escherichia coli RNase H hydrolysis of the RNA target only at high enzyme concentration.     

Binding of heparin to basic fibroblast growth factor induces a conformational change.

The binding of heparin to basic fibroblast growth factor (bFGF) induces a small but highly reproducible conformational change observable in the amide I region of the protein's infrared spectrum. The observed spectral changes suggest that the conformational change is highly localized most likely in the beta-turn regions of the bFGF molecule. Heparan sulfate, a component of the endothelial extracellular matrix, was also observed to bind to bFGF and induce a similar conformational change to that observed for heparin. Further, sucrose octasulfate, a compound which mimics the effects of heparin biologically, was also observed to induce this same conformational change. This spectroscopically observable change has allowed us to probe the functional determinants necessary for heparin to bind the bFGF and to induce the observed conformational change. We have determined the effects of binding of various monomeric and polymeric, sulfated and nonsulfated glycosaminoglycans and carbohydrate compounds. The results indicate that the binding of heparin involves highly specific interactions. Further, heparin was observed to greatly increase the thermal stability of bFGF, raising the Tm by 25 degrees C. Sucrose octasulfate was also able to enhance the thermal stability of bFGF, but not to the same extent as heparin.     

Characterization of ciprofloxacin binding to the linear single- and double-stranded DNA

The binding of ciprofloxacin to natural and synthetic polymeric DNAs was investigated at different solvent conditions using a combination of spectroscopic and hydrodynamic techniques. In 10 mM cacodylate buffer (pH 7.0) containing 108.6 mM Na(+), no sequence preferences in the interaction of ciprofloxacin with DNA was detected, while in 2 mM cacodylate buffer (pH 7.0) containing only 1.7 mM Na(+), a significant binding of ciprofloxacin to natural and synthetic linear double-stranded DNA was observed. At low ionic strength of solution, ciprofloxacin binding to DNA duplex containing alternating AT base pairs is accompanied by the largest enhancement in thermal stability (e.g. DeltaT(m) approximately 10 degrees C for poly[d(AT)].poly[d(AT)]), and the most pronounced red shift in the position of the maximum of the fluorescence emission spectrum (lambda(max)). Similar red shift in the position of lambda(max) is also observed for ciprofloxacin binding to dodecameric duplex containing five successive alternating AT base pairs in the row. On the other hand, ciprofloxacin binding to poly[d(GC)].poly[d(GC)], calf thymus DNA and dodecameric duplex containing a mixed sequence is accompanied by the largest fluorescence intensity quenching. Addition of NaCl does not completely displace ciprofloxacin bound to DNA, indicating the binding is not entirely electrostatic in origin. The intrinsic viscosity data suggest some degree of ciprofloxacin intercalation into duplex.     

A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs.

Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.     

The cystic fibrosis transmembrane conductance regulator. Effects of the most common cystic fibrosis-causing mutation on the secondary structure and stability of a synthetic peptide.

Deletion of phenylalanine 508 (delta Phe-508) in the cystic fibrosis transmembrane conductance regulator (CFTR) protein causes approximately 70% of all cases of cystic fibrosis. This residue lies in a region of the protein that we have synthesized chemically and shown to bind adenine nucleotides (Thomas, P. J., Shenbagamurthi, P., Ysern, X., and Pedersen, P. L. (1991) Science 251, 555-557). A peptide lacking this critical residue, but otherwise corresponding to this crucial part of the protein, now also has been chemically synthesized and purified. This mutant peptide (P-66) exhibits a significant loss of beta-sheet structure as compared with the wild type peptide (P-67). Furthermore, urea denaturation of peptide structure reveals that P-66 is less stable than P-67. Although under non-denaturing conditions both peptides bind adenine nucleotides with high affinity, the loss of structural stability is reflected in the binding function of the peptides. Thus, P-67, in contrast to P-66, retains a significant capacity for nucleotide binding in 4 M urea. These results suggest a model for impaired delta Phe-508 CFTR function.     

Thermodynamic stability of HLA-B*2705. Peptide complexes. Effect of peptide and major histocompatibility complex protein mutations

Designing synthetic vaccines from class I major histocompatibility complex (MHC)-binding antigenic peptides requires not only knowledge of the binding affinity of the designed peptide but also predicting the stability of the formed MHC-peptide complex. In order to better investigate structure-stability relationships, we have determined by circular dichroism spectroscopy the thermal stability of a class I MHC protein, HLA-B*2705, in complex with a set of 39 singly substituted peptide analogues. The influence of two anchoring side chains (P3 and P9) was studied by peptide mutation and appropriate site-directed mutagenesis of the HLA-B*2705 binding groove. The side chain at P9 is clearly the one that contributes the most to the thermal stability of the MHC-peptide complexes, as destabilization up to 25 degrees C are obtained after P9 mutation. Interestingly, structure-stability relationships do not fully mirror structure-binding relationships. As important as the C-terminal side chain are the terminal ammonium and carboxylate groups. Removal of a single H-bond between HLA-B27 and the terminal peptide moieties results in thermal destabilization up to 10 degrees C. Depending on the bound peptide and the location of the deleted H-bond, the decrease in the thermal stability of the corresponding complex is quantitatively different. The present study suggests that any peptidic amino acid at positions 3 and 9 promotes refolding of the B27-peptide complex. Once the complex is formed, the C-terminal side chain seems to play an important role for maintaining a stable complex.     

Effect of calcium and phosphatidic acid binding on the C2 domain of PKC alpha as studied by Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural and thermal denaturation of the C2 domain of PKC alpha (PKC-C2) and its complexes with Ca(2+) and phosphatidic acid vesicles. The amide I regions in the original spectra of PKC-C2 in the Ca(2+)-free and Ca(2+)-bound states are both consistent with a predominantly beta-sheet secondary structure below the denaturation temperatures. Spectroscopic studies of the thermal denaturation revealed that for the PKC-C2 domain alone the secondary structure abruptly changed at 50 degrees C. While in the presence of 2 and 12.5 mM Ca(2+), the thermal stability of the protein increased to 60 and 70 degrees C, respectively. Further studies using a mutant lacking two important amino acids involved in Ca(2+) binding (PKC-C2D246/248N) demonstrated that these mutations were inherently more stable to thermal denaturation than the wild-type protein. Phosphatidic acid binding to the PKC-C2 domain was characterized, and the lipid-protein binding became Ca(2+)-independent when 100 mol% phosphatidic acid vesicles were used. The mutant lacking two Ca(2+) binding sites was also able to bind to phosphatidic acid vesicles. The effect of lipid binding on secondary structure and thermal stability was also studied. Beta-sheet was the predominant structure observed in the lipid-bound state, although the percentage represented by this structure in the total area of the amide I band significantly decreased from 60% in the lipid-free state to 47% in the lipid-bound state. This decrease in the beta-sheet component of the lipid-bound complex correlates well with the significant increase observed in the 1644 cm(-1) band which can be assigned to loops and disordered structure. Thermal stability after lipid binding was very high, and no sign of thermal denaturation was observed in the presence of lipids under the conditions that were studied.     

Different Effects of Nonintercalative Antitumor Drugs on DNA Triple Helix Stability: SN-18071 Promotes Triple Helix Formation

Abstract

The interaction of the nonintercalating bisquaternary ammonium heterocyclic drugs SN- 18071 and SN-6999 with a DNA triple helix has been studied using thermal denaturation and CD spectroscopy. Our data show, that both minor groove binders can bind to the triple helix of poly(dA)-2poly(dT) under comparable ionic conditions, but they influence the stability of the triplex relative to the duplex structure of poly(dA)-poly(dT) in a different manner. SN- 18071, a ligand devoid of forming hydrogen bonds, can promote triplex formation and thermally stabilizes it up to 500 mM Na+ concentration. SN-6999 destabilizes the triplex to duplex equibilirium whereas it stabilizes the duplex. The binding constant of SN-18071 is found to be greater than that to the duplex. The stabilizing effect of SN-18071 is explained by electrostatic inetractions of three ligand molecules with the three grooves of the triple stranded structure. From the experiments it is concluded that SN-6999 binds to the triplex minor groove thereby destabilizing the triplex similar as previously reported for netropsin.     

The thermodynamics of drug-DNA interactions: ethidium bromide and propidium iodide

We report the first calorimetrically-derived characterization of the thermodynamics of ethidium bromide (EB) and propidium iodide (PI) binding to a series of nucleic acid host duplexes. Our spectroscopic and calorimetric measurements yield the following results: 1) At low salt (16mM Na+) and 25 degrees C. PI binds more strongly than EB to a given host duplex. The magnitude of this PI preference depends only marginally on base sequence, with AT base pairs showing a greater PI preference than GC base pairs. 2) The enhanced binding of PI relative to EB at low salt and 25 degrees C reflects a more favorable entropic driving force for PI binding. 3) The PI binding preference diminishes at higher salt concentrations (216mM). In other words, the binding preference is electrostatic in origin. 4) The salt dependence of the binding constants (delta lnKb/delta ln[Na+]) reveal that PI binds as a dication while EB binds as a monocation. 5) PI and EB both exhibit impressive enthalpy-entropy compensations when they bind to the deoxy homopolymers poly dA.poly dT and poly dA.poly dU. We have observed a similar enthalpy-entropy compensation for netropsin binding to the poly dA.poly dT homopolymer duplex. We therefore conclude that the compensation phenomenon is an intrinsic property of the host duplex rather than reflecting a property of the binding ligand. 6) When either PI or EB bind to the corresponding ribo homopolymer (poly rA.poy rU) we do not observe the enthalpy-entropy compensation that characterizes the binding to the deoxy homopolymer. 7) EB and PI both bind more strongly to poly d(AT).poly d(AT) than to poly d(AU).poly d(AU). Specifically, the absence of the thymine methyl group in poly d(AU).poly d(AU) reduces the binding constant of both drugs by a factor of four. This reduction in binding is due to a less favorable entropy change. In this paper we present and discuss possible molecular origins for our observed thermodynamic and extra-thermodynamic data. In particular, we evoke solvent effects involving both the drugs and the host duplexes when we propose molecular interpretations which are consistent with our thermodynamic data.     

RNA binding assays for Tat-derived peptides: implications for specificity.

RNA recognition by the HIV Tat protein is mediated in part by an arginine- and lysine-rich basic subdomain implicated as a signature element in proteins that bind RNA. Relative RNA binding affinities for a 14-residue peptide derived from Tat that spans the basic region are determined using a competition protocol. Binding specificity is compared with complexation by a 38-residue model for the RNA binding domain of Tat using the same approach. Binding strength for the minimal (14 residue) peptide is correlated with that for the longer peptide: both peptides recognize a short, bulged duplex. However, the shorter peptide dissociates more rapidly from the wild-type site and discriminates less well between nonspecific (double-stranded RNA) and specific sites. Relative dissociation constants for 38-residue peptide determined from direct partition and competition assays differ; the former assay consistently predicts stronger discrimination against RNAs with mutations in the stems flanking the bulge. Differences between the two assays are reconciled in terms of contributions from labile binding which is unstable to native gel electrophoresis. Kinetic stability may constitute a major specificity determinant for basic subdomain-mediated recognition of RNA.     

Replication protein A (RPA) binding to duplex cisplatin-damaged DNA is mediated through the generation of single-stranded DNA.

Replication protein A (RPA) is a heterotrimeric protein composed of 70-, 34-, and 14-kDa subunits that has been shown to be required for DNA replication, repair, and homologous recombination. We have previously shown preferential binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA compared with the control undamaged DNA (Patrick, S. M., and Turchi, J. J. (1998) Biochemistry 37, 8808-8815). Here we assess the binding of rhRPA to DNA containing site-specific cisplatin-DNA adducts. rhRPA is shown to bind 1.5-2-fold better to a duplex 30-base pair substrate containing a single 1,3d(GpXpG) compared with a 1,2d(GpG) cisplatin-DNA intrastrand adduct, consistent with the difference in thermal stability of DNA containing each adduct. Consistent with these data, a 21-base pair DNA substrate containing a centrally located single interstrand cisplatin cross-link resulted in less binding than to the undamaged control DNA. A series of experiments measuring rhRPA binding and concurrent DNA denaturation revealed that rhRPA binds duplex cisplatin-damaged DNA via the generation of single-stranded DNA. Single-strand DNA binding experiments show that rhRPA binds 3-4-fold better to an undamaged 24-base DNA compared with the same substrate containing a single 1,2d(GpG) cisplatin-DNA adduct. These data are consistent with a low affinity interaction of rhRPA with duplex-damaged DNA followed by the generation of single-stranded DNA and then high affinity binding to the undamaged DNA strand.     

Synthesis of oligonucleotide derivatives containing a bis-pyrene residue in the main chain.

The oligonucleotide having the bis-pyrene residue in the main chain was synthesized. The preparation of the bis-pyrene was started from the conversion of 2,2-bis-(bromomethyl)-1,3-propanediol into the protected bis-amino derivative. The reaction of the bis-amino derivative with 1-pyrenebutyric acid using DCC/HOBT afforded the desired bis-pyrene. This compound was then converted to the protected phosphormidite. The oligonucleotides possessing the bis-pyrene were synthesized by using the amidite. The oligonucleotides having the bis-pyrene residue can bind to DNA sequence in an aqueous solution to give the duplex with comparable thermal stability as that of the unmodified DNA/DNA duplex. The significantly enhanced pyrene-excimer fluorescence was observed upon hybridization of the bis-pyrene modified oligonucleotides with DNA.     

Characterization of a synthetic peptide that inhibits the interaction between protein S and C4b-binding protein

Protein S is unique among the vitamin K-dependent proteins found in blood plasma because it is a cofactor rather than a zymogen of a serine protease. Instead of a trypsin-like domain, protein S contains a domain that has sequence homology with steroid binding proteins. In order to understand the function of this structural domain, peptides have been synthesized with amino acid sequences that are homologous between human protein S and rat androgen binding protein. Two peptides, corresponding to amino acids 400-407 (PINPRLDG) and 605-614 (GVQLDLDEAI) of the protein S sequence have been tested for their effects on protein S function. Neither peptide altered the clotting of bovine or human plasma. The peptide GVQLDLDEAI enhanced the anticoagulant activity of human-activated protein C in human plasma while the peptide PINPRLDG had no effect. The peptide GVQLDLDEAI was observed to inhibit the binding of protein S to C4b-binding protein in plasma, resulting in increased concentrations of free protein S. GVQLDLDEAI was also observed to enhance the disassociation of the protein S.C4b-binding protein complex when purified complex was used. Finally, C4b-binding protein was observed to bind to GVQLDLDEAI. These results suggest that the carboxyl-terminal region of protein S, which contains the sequence GVQLDLDEAI, is involved in the interaction between protein S and C4b-binding protein.