NikR repressor: high-affinity nickel binding to the C-terminal domain regulates binding to operator DNA

E. coli NikR repressor binds operator DNA in a nickel-dependent fashion. The pM affinity of NikR for nickel is mediated by its C-terminal 86 residues. Nickel binding induced additional secondary structure, decreased the compactness, and increased the stability of NikR. Tetramer formation by the C-terminal domain and intact NikR did not require nickel. High-affinity nickel binding decreased the NikR concentration needed to half maximally protect operator DNA from undetectable levels to 30 nM. The intracellular concentration of NikR in E. coli is high enough that saturation of the high-affinity nickel sites should lead to substantial occupancy of the nik operator. Nickel binding to a set of low-affinity NikR sites resulted in an additional large increase in operator affinity and substantially increased the size of the NikR footprint on the operator.     

A synthetic miniprotein that binds specific DNA sequences by contacting both the major and the minor groove

Attachment of a slightly modified basic region of a bZIP protein (GCN4) to a distamycin-related tripyrrole provides a bivalent system capable of binding with high affinity to specific DNA sequences. Appropriate adjustment of the linker between the two units has led to a hybrid that binds a 9 base-pair-long DNA site (TTTTATGAC) with low nanomolar affinity at 4 degrees C. Circular dichroism and gel retardation studies indicate that the binding occurs by simultaneous insertion of the bZIP basic region into the DNA major groove and the tripyrrole moiety into the minor groove of the flanking sequence. Analysis of hybrids bearing alternative linkers revealed that tight, specific binding is strongly dependent on the length and nature of the connecting unit.     

Biosynthesis of a site-specific DNA cleaving protein

An E. coli catabolite activator protein (CAP) has been converted into a sequence-specific DNA cleaving protein by genetically introducing (2,2'-bipyridin-5-yl)alanine (Bpy-Ala) into the protein. The mutant CAP (CAP-K26Bpy-Ala) showed comparable binding affinity to CAP-WT for the consensus operator sequence. In the presence of Cu(II) and 3-mercaptopropionic acid, CAP-K26Bpy-Ala cleaves double-stranded DNA with high sequence specificity. This method should provide a useful tool for mapping the molecular details of protein-nucleic acid interactions.     

New computational strategy to analyze the interactions of ERalpha and ERbeta with different ERE sequences

The importance of computational methods for the simulation and analysis of biological systems has increased during the last years. In particular, methods to predict binding energies are developing not only with the aim of ranking the affinities between two or more complexes, but also to quantify the contribution of different types of interaction. In this work, we present the application of HINT, a non Newtonian force field, to rank the affinities of complexes formed by estrogen receptors (ER) alpha and beta and different estrogen responsive elements (ERE) near the estrogen-regulated genes. We used the crystallographic coordinates of the DNA binding domain of ERalpha complexed to a consensus ERE as a starting point to simulate several complexes in which some nucleotides in the ERE sequence were mutated. Moreover, we used homology modeling methods to create the structure of the complexes between the DNA binding domain of ERbeta (for which no experimental structures are currently available) and the same ERE sequences. Our results show that HINT is able to rank the affinities of ERalpha and ERbeta for different ERE sequences, and to correctly identify the positions on the DNA sequence that are most important for binding affinity. Moreover, the HINT output gives us the opportunity to identify and quantify the role played by each single atom of amino acids and nucleotides in the binding event, as well as to predict the effect on the binding affinity for other nucleotide mutations.     

Water-soluble amino derivatives of free-base dppz--syntheses and DNA binding studies

The syntheses of two water soluble dipyrido-[3,2-a:2',3'-c]-phenazine analogues containing one or two appended amino/amide chains are reported. Steady state optical studies on the two new compounds reveal high-energy dppz-based luminescence in water and non-aqueous solvents. Optical titrations with duplex DNA show that the luminescence is quenched on the addition of DNA. Binding curves constructed from absorption and emission changes indicate that, while one of the compounds display negligible binding properties, the other binds DNA with relatively high affinity (>10(5) M(-1)). Isothermal calorimetry experiments, designed to investigate the higher binding compound in more detail, reveals that its interaction with CT-DNA is actually biphasic with one tight (>10(5) M(-1)) and one weaker binding site (~10(5) M(-1)). In both cases binding is entropically driven. Further calorimetry studies involving the interaction of the new compound with a variety of polynucleotides were carried out. To aid comparisons, similar experiments involving a previously reported bipyridyldiylium derivative of dppz were also carried out. These studies reveal that the bipyridyldiylium derivative binds all these sequences monophasically with relatively low affinities (~10(4) M(-1)). However, while the amino/amide chain appended derivative binds to Poly(dA).poly(dT) monophasically with relatively low affinities, it binds all the other polynucleotide studied biphasically, with affinities ranging from <10(6) M(-1) to >10(8) M(-1). The ITC data reveals that for both compounds thermodynamic signatures for binding are dependent on the sequence being bound. In both cases, the data for Poly(dA).poly(dT) is particularly anomalous. An analysis of the data shows that binding is selective, with affinities at flexible sequences being several orders of magnitude higher than those at more rigid sequences.     

Use of Capillary Electrophoresis in the Study of Interaction between dsDNA and Drug Molecules

The advantages of berberine such as the anticancer1, antiinflammatory2 and no side effects of camptothecin1, have promoted the research in the mechanism of berberine with macrobiomolecules. In general, three different points of view have been presented on…     

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.     

Selection of aptamers for signal transduction proteins by capillary electrophoresis

High‐affinity aptamers for important signal transduction proteins, i.e. Cdc42‐GTP, p21‐activated kinase1 (PAK1) and MRCK (myotonic dystrophy kinase‐related Cdc42‐binding kinase) α were successfully selected in the low micro‐ to nanomolar range using non‐systematic evolution of ligands by exponential enrichment (SELEX) with at least three orders of magnitude enhancement from their respective bulk affinity of naïve DNA library. In the non‐SELEX procedure, CE was used as a highly efficient affinity method to select aptamers for the desired molecular target through a process that involved repetitive steps of partitioning, known as non‐equilibrium CE of equilibrium mixtures with no PCR amplification between successive steps. Various non‐SELEX conditions including the type, concentration and pH of the run buffer were optimized. Other considerations such as salt composition of selection buffer, protein concentration and sample injection size were also studied for high stringency during selection. After identifying the best enriched aptamer pool, randomly selected clones from the aptamer pool were sequenced to obtain the individual DNA sequences. The dissociation constants (K_d) of these sequences were in the low micromolar to nanomolar range, indicating high affinity to the respective proteins. The best binders were also subjected to sequence alignment to generate a phylogenetic tree. No significant consensus region based on approximately 50 sequences for each protein was observed, suggesting the high efficiency of non‐SELEX for the selection of numerous unique sequences with high selectivity.     

An Octahedral Cobalt(III) Complex with Axial NH3 Ligands that Templates and Selectively Stabilises G-quadruplex DNA

Guanine-rich sequences of DNA are known to readily fold into tetra-stranded helical structures known as G-quadruplexes (G4). Due to their biological relevance, G4s are potential anticancer drug targets and therefore there is significant interest in molecules with high affinity for these structures. Most G4 binders are polyaromatic planar compounds which π–π stack on the G4′s guanine tetrad. However, many of these compounds are not very selective since they can also intercalate into duplex DNA. Herein we report a new class of binder based on an octahedral cobalt(III) complex that binds to G4 via a different mode involving hydrogen bonding, electrostatic interactions and π–π stacking. We show that this new compound binds selectivity to G4 over duplex DNA (particularly to the G-rich sequence of the c-myc promoter). This new octahedral complex also has the ability to template the formation of G4 DNA from the unfolded sequence. Finally, we show that upon binding to G4, the complex prevents helicase Pif1-p from unfolding the c-myc G4 structure.     

Extending the language of DNA molecular recognition by polyamides: unexpected influence of imidazole and pyrrole arrangement on binding affinity and specificity

Pyrrole (Py) and imidazole (Im) polyamides can be designed to target specific DNA sequences. The effect that the pyrrole and imidazole arrangement, plus DNA sequence, have on sequence specificity and binding affinity has been investigated using DNA melting (DeltaT(M)), circular dichroism (CD), and surface plasmon resonance (SPR) studies. SPR results obtained from a complete set of triheterocyclic polyamides show a dramatic difference in the affinity of f-ImPyIm for its cognate DNA (K(eq) = 1.9 x 10(8) M(-1)) and f-PyPyIm for its cognate DNA (K(eq) = 5.9 x 10(5) M(-1)), which could not have been anticipated prior to characterization of these compounds. Moreover, f-ImPyIm has a 10-fold greater affinity for CGCG than distamycin A has for its cognate, AATT. To understand this difference, the triamide dimers are divided into two structural groupings: central and terminal pairings. The four possible central pairings show decreasing selectivity and affinity for their respective cognate sequences: -ImPy > -PyPy- > -PyIm- approximately -ImIm-. These results extend the language of current design motifs for polyamide sequence recognition to include the use of "words" for recognizing two adjacent base pairs, rather than "letters" for binding to single base pairs. Thus, polyamides designed to target Watson-Crick base pairs should utilize the strength of -ImPy- and -PyPy- central pairings. The f/Im and f/Py terminal groups yielded no advantage for their respective C/G or T/A base pairs. The exception is with the -ImPy- central pairing, for which f/Im has a 10-fold greater affinity for C/G than f/Py has for T/A.     

Small Sequence-Sensitive Compounds for Specific Recognition of the G⋅C Base Pair in DNA Minor Groove

A series of small diamidines with thiophene and modified N-alkylbenzimidazole σ-hole module represent specific binding to single G⋅C base pair (bp) DNA sequence. The variation of N-alkyl or aromatic rings were sensitive to microstructures of the DNA minor groove. Thirteen new compounds were synthesized to test their binding affinity and selectivity. The dicyanobenzimidazoles needed to synthesize the target diamidines were made via condensation/cyclization reactions of different aldehydes with different 3-amino-4-(alkyl- or phenyl-amino) benzonitriles. The final diamidines were synthesized using lithium bis-trimethylsilylamide (LiN[Si(CH₃)₃]₂) or Pinner methods. The newly synthesized compounds showed strong binding and selectivity to AAAGTTT compared to similar sequences AAATTT and AAAGCTTT investigated by several biophysical methods including biosensor-SPR, fluorescence spectroscopy, DNA thermal melting, ESI-MS spectrometry, circular dichroism, and molecular dynamics. The binding affinity results determined by fluorescence spectroscopy are in accordance with those obtained by biosensor-SPR. These small size single G⋅C bp highly specific binders extend the compound database for future biological applications.     

Investigation of non-covalent complexes of HIV-1 promoter DNA and polyamides containing N-methylpyrrole by electrospray ionization mass spectrometry

Eight novel polyamides containing N-methylpyrrole were designed to target the sequence (5'-CTGCATATAAGCAG-3'/5'-CTGCTTATATGCAG-3') of the TATA box element of the HIV-1 promoter DNA. The non-covalent complexes of the promoter DNA and the polyamides were investigated by electrospray ionization (ESI) mass spectrometry, which provided strong evidence for the binding of the novel polyamides to the sequence of the TATA box element. It also revealed that polyamide 2 (PyPyPyPybetaDp), a potent binder of HIV-1 promoter DNA and a lead molecule for the design of new anti-HIV-1 drugs, had the highest binding affinity with the TATA box element DNA among these polyamides by examining the stoichiometry and the selectivity.     

Sequence‐Specific DNA Recognition by Cyclic Pyrrole–Imidazole Cysteine‐Derived Polyamide Dimers

Pyrrole–imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence‐specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence‐specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β‐alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA.     

Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein

Background

Single-stranded DNA binding proteins (SSB) are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase.

Results

We have cloned and purified SSB from Bacillus anthracis (SSB_BA). In the absence of DNA, at concentrations ??100 ??g/ml, SSB_BA did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSB_BA bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT)₇₀ binding suggested that SSB_BA forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure.

Conclusions

Unlike other prokaryotic SSB proteins, SSB_BA from Bacillus anthracis appeared to be monomeric at concentrations ??100 ??g/ml as determined by SE-HPLC. SSB_BA retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSB_BA appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSB_BA could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication.     

Heterocyclic diamidine interactions at AT base pairs in the DNA minor groove: effects of heterocycle differences, DNA AT sequence and length

Given the increasing significance of diamidines as DNA-targeted therapeutics and biotechnology reagents, it is important to establish the variations in thermodynamic quantities that characterize the interactions of closely related compounds to different sequence AT binding sites. In this study, an array of methods including biosensor-surface plasmon resonance (SPR), isothermal titration microcalorimetry (ITC), circular dichroism (CD), thermal melting (Tm) and molecular modeling have been used to characterize the binding of dicationic diamidines related to DB75 (amidine-phenyl-furan-phenyl-amidine) with alternating and nonalternating AT sequences. Conversion of the central furan of DB75 to other similar groups, such as thiophene or selenophene, can yield compounds with increased affinity and sequence binding selectivity for the minor groove. Calorimetric measurements revealed that the thermodynamic parameters (Delta G, Delta H, Delta S) that drive diamidine binding to alternating and nonalternating oligomers can be quite different and depend on both DNA sequence and length. Small changes in a compound can have major effects on DNA interactions. By choosing an appropriate central group it is possible to "tune" the shape of the molecule to match DNA for enhanced affinity and sequence recognition.