The effects of local DNA sequence on the interaction of ligands with their preferred binding sites

We have examined the effects of local DNA sequence on the interaction of distamycin, Hoechst 33258, echinomycin, actinomycin and mithramycin with their preferred binding sites using a series of DNA fragments that contain every symmetrical hexanucleotide sequence. In several instances we find that the affinity for the ligands' preferred binding sites is affected by the hexanucleotide context in which they are located. The AT-selective minor groove binding ligand Hoechst 33258 shows a 200-fold difference in binding to the 16 different X(A/T)(4)Y sites; the strongest binding is to AAATTT and the weakest is to (G/C)TTAA(C/G). Although TTAA is generally a poor binding site, ATTAAT is better than TTTAAA and they are both much better than GTTAAC and CTTAAG. Similarly, TTATAA and ATATAT are better binding sites than GTATAC and CTATAG. In contrast, distamycin shows less discrimination between the various X(A/T)(4)Y sites, with a 20-fold difference between the best [(A/T)AATT(T/A)] and worst [GATATC and (G/C)TTAA(C/G)] sites. Although actinomycin binds to GpC it shows little or no interaction with any of the GGCC sites, yet shows only a six-fold variation in affinities for the other XYGCXY sites. Echinomycin binds to CpG yet shows no binding to TTCGAA, TGCGCA and AGCGCT, while the best binding is to AACGTT. The tetranucleotides CCGG and ACGT produce consistently good binding sites, irrespective of the surrounding sequences, while the interaction with TCGA and GCGC is sensitive to the hexanucleotide context. Hexanucleotides with a central GCGC, flanked by A and T are weaker echinomycin sites than those flanked by G and C, especially CGCGCG. The best X(G/C)(4)Y binding sites for mithramycin were located at AGCGCT and GGGCCC, and the worst at CCCGGG and TCCGGA. These footprinting fragments are valuable tools for comparing the binding of ligands to all the potential symmetrical hexanucleotides and provide insights into the effects of local DNA sequence on ligand-DNA interactions.     

Combining nucleoside analogues to achieve recognition of oligopurine tracts by triplex-forming oligonucleotides at physiological pH

We have used DNase I footprinting to examine DNA triple helix formation at a 12 base pair oligopurine.oligopyrimidine sequence, using oligonucleotides that contain combinations of 2'-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine (bis-amino-U, BAU) and 3-methyl-2-aminopyridine (MeP) in place of T and C, respectively. This combination acts cooperatively to enable high affinity triple helix formation at physiological pH. The affinity depends on the number of substitutions and their arrangement; oligonucleotides in which these analogues are evenly distributed throughout the third strand bind much better than those in which they are clustered together.     

DNA recognition by DNase I

Bovine pancreatic DNase I shows a strong preference for double-stranded substrates and cleaves DNA with strongly varying cutting rates suggesting that the enzyme recognises sequence-dependent structural variations of the DNA double helix. The complicated cleavage pattern indicates that several local as well as global helix parameters influences the cutting frequency of DNase I at a given bond. The high resolution crystal structures of two DNase I-DNA complexes showed that the enzyme binds tightly in the minor groove, and to the sugar-phosphate backbones of both strands, and thereby induces a widening of the minor groove and a bending towards the major grooves. In agreement with biochemical data this suggests that flexibility and minor groove geometry are major parameters determining the cutting rate of DNase I. Experimental observations showing that the sequence environmental of a dinucleotide step strongly affects its cleavage efficiency can be rationalized by that fact that six base pair are in contact with the enzyme. Mutational analysis based on the structural results has identified critical residues for DNA binding and cleavage and has lead to a proposal for the catalytic mechanism.     

Specific recognition of loop DNA by dicationic porphyrins

A series of dicationic porphyrins were found to specifically recognize loop structures of oligodeoxynucleotides, and to selectively oxidize guanine residues upon photo-irradiation at micromolar concentrations. These compounds could, thus, be employed as promising structural probes for specific secondary DNA structures. The production of singlet oxygen ((1)O2) is responsible for the DNA-modification profiles. Both UV-titration and thermal-melting experiments indicate that the strong affinity of the charged porphyrins towards DNA loops is responsible for these molecular-recognition phenomena.     

Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD

Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of alpha-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes.     

Molecular recognition of a DNA:RNA hybrid: sub-nanomolar binding by a neomycin-methidium conjugate

A novel neomycin–methidium conjugate was synthesized. The covalent linkage of the aminoglycoside to an intercalator, a derivative of ethidium bromide, results in a new conjugate capable of selective recognition of the DNA:RNA hybrid duplex. Spectroscopic methods: UV, CD, fluorescence, and calorimetric techniques: DSC and ITC were used to characterize the sub-nanomolar binding displayed by the conjugate for the DNA:RNA hybrid duplex, poly(dA):poly(rU).     

Specificity in DNA recognition by phage integrases

The λ-related (lambdoid) coliphages are related to one another by frequent natural recombination and maintain a high level of functional polymorphism for several activities of the phages. Arguments are presented that the polymorphism of the integration module results from selection (presumably frequency-dependent) for new (not improved) specificities of site recognition. Analysis of phages λ and HK022 by Weisberg and collaborators previously showed that changes in five noncontiguous amino acids could switch site recognition specificity. Phage 21 and defective element e14, which integrate at the same site, differ in recognition specificity for both core and arm sites. In vitro assays of e14 and 21 insertion and excision confirm this conclusion. Inhibition by ds arm site oligonucleotides defines the sequence specificity more precisely.     

Synthetic α-helical peptides incorporating intercalators for DNA recognition

The design and synthesis of a water-soluble 14-residue peptide, in which a quinoline intercalator is attached to the peptide backbone via alkylation of a central cysteine residue, is reported. 600 MHz ¹H NMR spectroscopy and circular dichroism indicate that the peptide forms a nascent helix in aqueous solution, ie. an ensemble of turn-like structures over several adjacent residues in the peptide. A large number of sequential d_NN(i, i+1) connectivities were observed in NOESY spectra, and titration of trifluoroethanol into a solution of the peptide resulted in the characteristic CD spectrum expected for an α-helix. At low DNA concentrations, CD spectroscopy indicates that this helical conformation is stabilized, presumably due to folding of the peptide in the major groove of DNA.     

DNA recognition and nucleosome organization

The affinity of a DNA sequence for the histone octamer in a core nucleosome depends on the intrinsic flexibility of the DNA. This parameter can be affected both by the sequence-dependent conformational preferences of individual base steps and by the nature and location of the exocyclic groups of the DNA bases. By adopting highly preferred conformations particular types of base step can influence the rotational positioning of the DNA on the surface of the histone octamer. The asymmetry of the next higher order of chromatin structure is determined in part by the asymmetric binding of the globular domain of histone H5 to the core nucleosome. © 1998 John Wiley & Sons, Inc. Biopoly 44: 423–433 1997     

Self-structure induction in single stranded poly(A) by small molecules: Studies on DNA intercalators, partial intercalators and groove binding molecules

Self-structure induction in single stranded poly(A) has been one typical example of the various ways that could be used to modulate nucleic acid structural aspects through binding of small molecules. For the first time, the interaction between a series of small molecules and poly(A) has been investigated to understand the nature of the structural features in DNA binding small molecules that could be responsible for the formation of self-structure in single stranded poly(A) molecules. Classical intercalators like ethidium, coralyne, quinacrine and proflavine, partial intercalators like berberine and palmatine and classical minor groove binders like hoechst 33258 and DAPI have been chosen for this study. The binding of each of these molecules to poly(A) has been characterized by absorption spectral titration, job plot and isothermal titration calorimetry. Self-structure formation was monitored from circular dichroic melting, optical melting and differential scanning calorimetry. The results revealed that while all the intercalators studied induced self-structure formation, partial intercalators did not induce the same in poly(A). Of the two classical DNA minor groove binding molecules investigated, hoechst was effective in inducing self-structure while DAPI was ineffective. Self-structure induction in poly(A) was observed to be directly linked to the cooperative binding of the molecules to poly(A) in that all the molecules that bound cooperatively induced self-structure in poly(A). Structural and thermodynamic aspects of the interaction leading to self-structure formation are described.     

Biomolecular recognition in DNA tagged CdSe nanowires

DNA template driven CdSe nanobeads (NBs) and nanowires (NWs) have been synthesized by an electrodeposition technique. The synthesis protocol has yielded randomly oriented cubic CdSe NBs with mean size 3.0 nm in presence of single stranded DNA, poly G(30). Monocrystalline cubic CdSe NWs of width 4.0 nm with string-like morphology have been achieved when synthesized in presence of both poly G(30) and its conjugate, poly C(30). Optical absorption of CdSe NBs show a blue shift of 0.8 eV and long wavelength tailing where as NWs show steep increase of absorption in shorter wavelength regime accompanied by a further blue shift. DNA tags to the NBs or NWs have been confirmed from Fourier transform infrared spectroscopy measurements. Biomolecular recognition with CdSe NWs have been established by photoluminescence measurements.     

DNA recognition by structure-selective nucleases

The nucleases discussed in this review show little sequence specificity but instead recognize certain structural features of their respective DNA substrates. The level of their structural selectivity ranges from simple discrimination between single- and double-stranded DNA (nucleases P1 and S1), the recognition of helical parameters like groove width and flexibility (DNase I), the recognition of helical distortions caused by abasic sites (exonuclease III, HAP1), to the recognition of specialized structures like flap DNA (5′-nucleases of eukaryotes, phages, and eubacterial DNA polymerases) and four-way junctions (T4 endonuclease VII, RuvC). The discussion is focused on the structural basis of the recognition process. In most cases the available x-ray structures of the nucleases and/or their DNA complexes have revealed the presence of structural motifs explaining the observed structural selectivity. © 1998 John Wiley & Sons, Inc. Biopoly 44: 405–421, 1997     

Specificity in DNA recognition by a peptide from papillomavirus E2 protein

The E2 proteins of papillomavirus specifically bind to double-stranded DNA containing the consensus sequence ACCG-N4-CGGT, where N is any nucleotide. Here, we show the binding and recognition of dissimilar DNA sequences by an 18 amino-acid peptide (alpha1E2), which corresponds to the DNA-recognition helix, alpha-helix-1. Isothermal DNA binding assays performed with the DNA consensus sequence show saturable curves with alpha1E2 peptide, and the alpha1E2 peptide is converted to an ordered conformation upon complexation. Measurements performed with non-specific DNA sequence fail to saturate, a behavior characteristic of non-specific binding. Binding of the alpha1E2 peptide to these DNA sequences display a different counter-ion dependence, indicating a dissimilar, sequence-dependent mechanism of interaction. Quantitative stoichiometric measurements revealed the specificity in alpha1E2 peptide recognition of the ACCG half-site, demonstrating capacity for discrimination of nucleic acid bases sequences without the need of a whole protein architecture.     

Role of base flipping in specific recognition of damaged DNA by repair enzymes

DNA repair enzymes induce base flipping in the process of damage recognition. Endonuclease V initiates the repair of cis, syn thymine dimers (TD) produced in DNA by UV radiation. The enzyme is known to flip the base opposite the damage into a non-specific binding pocket inside the protein. Uracil DNA glycosylase removes a uracil base from G.U mismatches in DNA by initially flipping it into a highly specific pocket in the enzyme. The contribution of base flipping to specific recognition has been studied by molecular dynamics simulations on the closed and open states of undamaged and damaged models of DNA. Analysis of the distributions of bending and opening angles indicates that enhanced base flipping originates in increased flexibility of the damaged DNA and the lowering of the energy difference between the closed and open states. The increased flexibility of the damaged DNA gives rise to a DNA more susceptible to distortions induced by the enzyme, which lowers the barrier for base flipping. The free energy profile of the base-flipping process was constructed using a potential of mean force representation. The barrier for TD-containing DNA is 2.5 kcal mol(-1) lower than that in the undamaged DNA, while the barrier for uracil flipping is 11.6 kcal mol(-1) lower than the barrier for flipping a cytosine base in the undamaged DNA. The final barriers for base flipping are approximately 10 kcal mol(-1), making the rate of base flipping similar to the rate of linear scanning of proteins on DNA. These results suggest that damage recognition based on lowering the barrier for base flipping can provide a general mechanism for other DNA-repair enzymes.     

The binding of actinomycin D and adriamycin to supercoiled DNA, single-stranded DNA and polynucleotides

The effect of actinomycin D and adriamycin on synthetic polynucleotides, single-stranded viral DNA and supercoiled DNA has been studied employing the fluorescent probe, terbium. Marked displacement of the probe was observed when any deoxyribose-containing polynucleotide was pretreated with either drug. With supercoiled DNA, an unwinding of the supercoil was observed at very low drug concentrations (at approx. 1:500 molar ratio of drug:DNA) prior to the displacement of the terbium. This unwinding was visualized by agarose gel electrophoresis at molar ratios of approx. 1:200. The effect was more apparent and occurred at lower drug:DNA ratios with actinomycin D than with adriamycin. Unlike cis-dichlorodiammine platinum(II), actinomycin D did not protect pBR322 DNA from cleavage at its BamHI site. The hydrolysis of Φχ174 DNA by a series of G-C-specific restriction nucleases (including HhaI, HpaII and HaeIII) was also not affected by prior treatment of the DNA with actinomycin D.