Design of peptide that recognizes double-stranded DNA.

A novel molecular tool for double-stranded (ds) DNA detection using synthetic peptide is described. The peptide was designed based on the DNA binding domain of the lambda phage CRO repressor (CRO). The designed peptides contain helix-turn-helix (HTH), which is DNA binding motif. A cyclic peptide and a mutant peptide based on CRO were also designed, and the resulting affinity for dsDNA was increased. Furthermore, native amino acids of the peptide were replaced with arginine to increase the affinity for dsDNA. The affinity of these peptides for DNA binding was assessed by surface plasmon resonance (SPR) technique.     

Photocontrol of DNA binding specificity of a miniature engrailed homeodomain

Control of DNA binding of HDH-3, a 18-residue polypeptide based on the recognition helix of the Q50K engrailed homeodomain, has been achieved. HDH-3 was linked to an azobenzene cross-linker through two cysteine residues in an i, i + 11 spacing. For the thermodynamically stable trans configuration of the cross-linker, the dark-adapted peptide (dad-HDH-3) adopted a mainly alpha-helical structure as judged by circular dichroism (CD) spectroscopy. After irradiation with light of 360 nm, the helical content of the peptide (irrad-HDH-3) was reduced significantly and the CD spectrum of the irradiated peptide resembled that of the largely unstructured, unalkylated peptide. Despite lacking helices-1 and -2 and the N-terminal arm of Q50K engrailed, dad-HDH-3 bound to its natural DNA target sequence TAATCC (QRE) with high affinity (K(D) = 7.5 +/- 1.3 nM). The binding affinity for the mutant DNA sequence, TAATTA (ERE), was reduced significantly (K(D) = 140 +/- 11 nM). Unlike irrad-HDH-3, which like the unalkylated parent peptide displayed only marginal DNA binding specificity, dad-HDH-3 specified base pairs 5 and 6 of QRE with an accuracy rivaling that of the intact wild-type Q50K engrailed homeodomain, making dad-HDH-3 the most specific designed DNA binding miniature homeodomain reported to date. Moreover, DNA binding affinity and specificity of HDH-3 could be controlled externally by irradiation with light.     

A general strategy to determine a target DNA sequence of a short peptide: application to a d-peptide.

Short peptides could potentially provide a novel element to read-out DNA sequences from the major groove. However, it is difficult to determine sequence-preference of de novo designed monomeric short peptides. Because DNS-binding affinity and specificity of short peptides are usually much lower than those of native DNA-binding proteins, determining the sequence-preference of short peptides by conventional methods utilized to deduce the target sequence of proteins often produces an unclear outcome. We report here a general strategy to defining the sequence-preference of a DNA-binding short peptide by using the heterodimers. A GCN4 basic region peptide tethers a low-affinity DNA-binding peptide adjacent to a GCN4 binding sequence through the cyclodextrin-adamantane association, thereby increasing local concentration of the low-affinity peptide on degenerated DNA sequences. An increase of the local concentration allows one to select a preferential sequence for the low-affinity DNA binding peptide. The method successfully identified specific sequences of short peptides derived from native DNA-binding proteins. The usefulness of this approach has been demonstrated by identifying preferred DNA targets for a peptide composed only of d-amino acids. The method is potentially applicable not only to artificial peptides, but also to other synthethic ligands.     

Controlling the DNA binding specificity of bHLH proteins through intramolecular interactions

Reversible control of the conformation of proteins was employed to probe the relationship between flexibility and specificity of the basic helix-loop-helix protein MyoD. A fusion protein (apaMyoD) was designed where the basic DNA binding helix of MyoD was stablized by an amino-terminal extension with a sequence derived from the bee venom peptide apamin. The disulfide-stabilized helix from apamin served as a nucleus for a helix that extended for a further ten residues, thereby holding apaMyoD's DNA recognition helix in a predominantly alpha-helical conformation. The thermal stability of the DNA complexes of apaMyoD was increased by 13 degrees C relative to MyoD-bHLH. Measurements of the fluorescence anisotropy change on DNA binding indicated that apaMyoD bound to E-box-containing DNA sequences with enhanced affinity relative to MyoD-bHLH. Consequently, the DNA binding specificity of apaMyoD was increased 10-fold.     

Sequence-specific binding of DNA to liposomes containing di-alkyl peptide nucleic acid (PNA) amphiphiles

We present a method to covalently attach peptide nucleic acid (PNA) to liposomes by conjugation of PNA peptide to charged amino acids and synthetic di-alkyl lipids ("PNA amphiphile," PNAA) followed by co-extrusion with disteroylphosphatidylcholine (DSPC) and cholesterol. Attachment of four Glu residues and two ethylene oxide spacers to the PNAA was required to confer proper hydration for extrusion and presentation for DNA hybridization. The extent of DNA oligomer binding to 10-mer PNAA liposomes was assessed using capillary zone electrophoresis. Nearly all PNAs on the liposome surface are complexed with a stoichiometric amount of complementary DNA 10-mers after 3-h incubation in pH 8.0 Tris buffer. No binding to PNAA liposomes was observed using DNA 10-mers with a single mismatch. Longer DNA showed a greatly attenuated binding efficiency, likely because of electrostatic repulsion between the PNAA liposome double layer and the DNA backbone. Langmuir isotherms of PNAA:DSPC:chol monolayers indicate miscibility of these components at the compositions used for liposome preparation. PNAA liposomes preserve the high sequence-selectivity of PNAs and emerge as a useful sequence tag for highly sensitive bioanalytical devices.     

Self-assembling poly( -lysine)/DNA complexes capable of integrin-mediated cellular uptake and gene expression

Integrin-mediated delivery of genes is evaluated using a synthetic vector formed by self-assembly of DNA with an oligolysine- peptide sequence containing RGD (referred to as K₁₆-RGD). The RGD peptide binds plasmid DNA effectively and inhibits ethidium bromide/DNA fluorescence at N-to-P ratios of less than 1.0. At N:P ratio 1.0, peptide/DNA complexes formed show a mixture of normal DNA migration and retention at the origin when analysed by agarose electrophoresis. At N:P ratio of 1.2, the complexes have a slight positive surface charge (5 mV) and in the absence of serum they show 10-fold increase uptake into 293 cells, compared with control poly( -lysine)/DNA vectors, together with a 100-fold increase in transfection. In the presence of serum, RGD-mediated uptake is decreased about 3-fold, but the targeted vectors achieve over 150 times greater transfection than poly( -lysine)/DNA controls. Transfection could be inhibited by addition of competing RGD, and to a lesser extent RGE, peptides. The targeted vector is believed to achieve cell uptake and transfection by binding av integrins in the cell surface, and the approach could be employed to promote internalisation of vectors following their binding to other, high affinity, receptors, in a system analogous to adenovirus entry.     

Superior duplex DNA strand invasion by acridine conjugated peptide nucleic acids

DNA helix invasion by P-loop forming peptide nucleic acids (PNAs) is extremely sensitive to increased ionic strength as this stabilizes the DNA duplex. To address this, the DNA intercalator 9-aminoacridine was conjugated to helix invading PNAs, and the duplex DNA binding efficiency of such constructs was measured at different ionic strength conditions by electrophoretic mobility shift analysis. Remarkably, at physiogically relevant ionic strength (140 mM K+/10 mM Na+, 2 mM Mg2+), acridine conjugated PNAs showed 20-150-fold superior binding to a cognate sequence target as compared to the conventional PNAs. This enhancement occurred without compromising the sequence specificity of binding. Thus, simply conjugating the DNA intercalator 9-aminoacridine to PNA represents a major step toward the development of helix invading constructs for in vivo applications such as gene targeting.     

DARK INTERACTION AND OXYGEN DEPENDENT PHOTOBINDING BETWEEN AN AROMATIC TETRAPEPTIDE AND DNA MODIFIED BY 5- METHOXYPSORALEN MONOADDUCTS

The binding of the tetrapeptide lysyl-tryptophyl-glycyl-lysine t -butyl ester to native DNA and DNA photochemically modified by 5-methoxypsoralen has been investigated by fluorescence spectroscopy. The peptide exhibits a higher affinity for the modified DNA than for the undamaged DNA. This is due to the presence of strong stacking sites in the vicinity of 5-methoxypsoralen monoadducts. Upon irradiation at 365 nm of the peptide-modified DNA complexes, a cross-linking of the peptide to the nucleic acid is observed. This photochemical addition requires the presence of oxygen and involves, in part, singlet oxygen.     

From IHF protein to design and synthesis of a sequence-specific DNA bending peptide

The design and synthesis of a small peptide that mimics the integration host factor (IHF), a major nucleoid-associated protein, is reported. IHF induces DNA compaction by sequence-specific binding that leads to significant bending of the DNA double strand. In a modular approach a small L-lysine dendrimer responsible for nonspecific charge-charge interactions was linked to a cyclopeptide. The latter was designed for specific DNA recognition in the minor groove followed by bending of the double strand.     

Towards sequence selective DNA binding: design, synthesis and DNA binding studies of novel bis-porphyrin peptidic nanostructures

A new series of peptidic nanostructures bearing two intercalating moieties was designed and synthesized to achieve selective recognition of DNA sequences. A cationic porphyrin was attached to a glutamic acid side chain and the latter introduced into a peptidic sequence by standard solid-phase peptide synthesis methodology. Conformation of the hydrosoluble peptidic structures bearing two cationic porphyrins was studied by circular dichroism. Using UV-visible spectroscopy and induced circular dichroism, we demonstrate that the compounds are fully intercalated upon binding to double-stranded DNA and that the compounds exhibit a tremendous preference for GC over AT sequences for intercalation.     

Peptide bis-intercalator binds DNA via threading mode with sequence specific contacts in the major groove

BACKGROUND: We previously described a general class of DNA polyintercalators in which 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) intercalating units are connected via peptide linkers, resulting in the first known tetrakis- and octakis-intercalators. We showed further that changes in the composition of the peptide tether result in novel DNA binding site specificities. We now examine in detail the DNA binding mode and sequence specific recognition of Compound 1, an NDI bis-intercalator containing the peptide linker gly-gly-gly-lys. RESULTS: 1H-NMR structural studies of Compound 1 bound to d(CGGTACCG)(2) confirmed a threading mode of intercalation, with four base pairs between the diimide units. The NMR data, combined with DNAse I footprinting of several analogs, suggest that specificity depends on a combination of steric and electrostatic contacts by the peptide linker in the floor of the major groove. CONCLUSIONS: In view of the modular nature and facile synthesis of our NDI-based polyintercalators, such structural knowledge can be used to improve or alter the specificity of the compounds and design longer polyintercalators that recognize correspondingly longer DNA sequences with alternating access to both DNA grooves.     

Direction control in DNA binding of chiral D-lysine-based peptide nucleic acid (PNA) probed by electrospray mass spectrometry

The DNA binding abilities of peptide nucleic acids (PNAs), both achiral and bearing three adjacent D-lysine-based monomers in the middle of the strand ("chiral box" PNA), were studied by means of electrospray mass spectrometry (ESI-MS). In contrast with achiral PNA, "Chiral box" PNA was confirmed to exert high direction control (antiparallel vs. parallel DNA target) in DNA binding.     

Screening of threading bis-intercalators binding to duplex DNA by electrospray ionization tandem mass spectrometry

The DNA binding of novel threading bis-intercalators V1, trans-D1, and cis-C1, which contain two naphthalene diimide (NDI) intercalation units connected by a scaffold, was evaluated using electrospray ionization mass spectrometry (ESI-MS) and DNAse footprinting techniques. ESI-MS experiments confirmed that V1, the ligand containing the -Gly3-Lys- peptide scaffold, binds to a DNA duplex containing the 5'-GGTACC-3' specific binding site identified in previous NMR-based studies. The ligand formed complexes with a ligand/DNA binding stoichiometry of 1:1, even when there was excess ligand in solution. Trans-D1 and cis-C1 are new ligands containing a rigid spiro-tricyclic scaffold in the trans- and cis- orientations, respectively. Preliminary DNAse footprinting experiments identified possible specific binding sites of 5'-CAGTGA-5' for trans-D1 and 5'-GGTACC-3' for cis-C1. ESI-MS experiments revealed that both ligands bound to DNA duplexes containing the respective specific binding sequences, with cis-C1 exhibiting the most extensive binding based on a higher fraction of bound DNA value. Cis-C1 formed complexes with a dominant 1:1 binding stoichiometry, whereas trans-D1 was able to form 2:1 complexes at ligand/DNA molar ratios >or=1 which is suggestive of nonspecific binding. Collisional activated dissociation (CAD) experiments indicate that DNA complexes containing V1, trans-D1, and cis-C1 have a unique fragmentation pathway, which was also observed for complexes containing the commercially available bis-intercalator echinomycin, as a result of similar binding interactions, marked by intercalation in addition to hydrogen bonding by the scaffold with the DNA major or minor groove.     

Studies of interaction between a new synthesized minor-groove targeting artificial nuclease and DNA

Nuclease plays an important role in molecular biology, such as DNA sequencing. Synthetic polyamide conjugates can be considered as new tool in the selective inhibition of gene expression and as potential drugs in anticancer or antiviral chemotherapy. In this paper, a new synthesized minor-groove targeting artificial nuclease, oligopyrrol-containing peptide, was reported. It was found that this new compound can bind DNA in AT-riched minor groove with high affinity and site specificity. DNA binding behavior was determined by UV-vis and circular dichroism (CD) methods. It was indicated that compound 6 can enhance the Tm of oligomer DNA from 51.8 to 63.5 degrees C and possesses large binding constant (Kb=8.83x10(4)L/mol).     

NMR structural analysis of a modular threading tetraintercalator bound to DNA

The synthesis and NMR structural studies are reported for a modular threading tetraintercalator bound to DNA. The tetraintercalator design is based on 1,4,5,8-tetracarboxylic naphthalene diimide units connected through flexible peptide linkers. Aided by an overall C(2) symmetry, NMR analysis verified a threading polyintercalation mode of binding, with linkers alternating in the order minor groove, major groove, minor groove, analogous to how a snake might climb a ladder. This study represents the first NMR analysis of a threading tetraintercalator and, as such, structurally characterizes a new topology for molecules that bind to relatively long DNA sequences with extensive access to both DNA grooves.     

NETROPSIN: INTERACTION WITH ULTRAVIOLET IRRADIATED DNA

Abstract—Ultraviolet irradiation of double-stranded DNA reduces the circular dichroism (i < 300 nm) induced when the basic peptide antibiotic netropsin (Nt) is added to DNA subsequent to thc irradiation compared to the CD induced by the same concentrations of Nt added to unirradiated DNA. Nt is known to bind to A T base pairs in duplex DNA but will not bind to single-stranded DNA. The reduction in the maximum induced CD observed with saturating concentrations of Nt is a linear function of the concentration of pyrimidine dimers which. along with other dinucleotide photoproducts. form short disrupted regions in duplex DNA. The decrease in the CD of Nt bound to irradiated DNA could be due to elimination of potential Nt sites in the vicinity of a dimer. reduction in the average magnitude of the CD of Nt bound near a dimer or various combinations of these effects. In addition there is a reduction in the average binding constant for Nt bound to irradiated DNA compared to unirradiated DNA suggesting that formation of dinucleotide photoproducts either tends to preferentially eliminate the tighter binding sites or that tighter sites are converted to weaker ones. A simple model suggests that no more than one-third to one-half of the pyrimidine dimcrs formed in DNA completely eliminate a Nt site.     

Single-nucleotide-specific PNA-peptide ligation on synthetic and PCR DNA templates

DNA-directed chemical synthesis has matured into a useful tool with applications such as fabrication of defined (nano)molecular architectures, evolution of amplifiable small-molecule libraries, and nucleic acid detection. Most commonly, chemical methods were used to join oligonucleotides under the control of a DNA or RNA template. The full potential of chemical ligation reactions can be uncovered when nonnatural oligonucleotide analogues that can provide new opportunities such as increased stability, DNA affinity, hybridization selectivity, and/or ease and accuracy of detection are employed. It is shown that peptide nucleic acid (PNA) conjugates, nonionic biostable DNA analogues, allowed the fashioning of highly chemoselective and sequence-selective peptide ligation methods. In particular, PNA-mediated native chemical ligations proceed with sequence selectivities and ligation rates that reach those of ligase-catalyzed oligodeoxynucleotide reactions. Usually, sequence-specific ligations can only be achieved by employing short-length probes, which show DNA affinities that are too low to allow stable binding to target segments in large, double-stranded DNA. It is demonstrated that the PNA-based ligation chemistry allowed the development of a homogeneous system in which rapid single-base mutation analyses can be performed even on double-stranded PCR DNA templates.