DNA binding of an organic dppz-based intercalator

An improved synthesis of a water-soluble derivative of dipyrido[3,2-a:2',3'-c]phenazine (dppz) is reported. The structures of both dppz and the cation ethylene-bipyridyldiylium-phenazine dinitrate [[1][(PF(6))(2)]] have been obtained via X-ray crystallography. Metal complex derivatives of dppz are very well studied. However, using the water soluble [1][(NO(3))(2)], the nature of the interaction of a simple dppz unit with duplex DNA has been investigated for the first time. In both organic solvents and water, 1 displays unstructured luminescence, assigned to an intramolecular charge transfer. The emission is quenched on binding to natural and synthetic duplex DNA, including poly(dA).poly(dT). A variety of techniques reveal that the cation binds to DNA with an affinity comparable to those of many metal dppz complexes, via an intercalative binding mode.     

High-resolution assessment of protein DNA binding affinity and selectivity utilizing a fluorescent intercalator displacement (FID) assay

Protein titration displacement of ethidium bromide bound to hairpin deoxyoligonucleotides containing any sequence of interest provides a well-defined titration curve (measuring the loss of fluorescence derived from the DNA bound ethidium bromide) that provides both absolute binding constants (K(a)) and stoichiometry of binding. This use of a fluorescent intercalator displacement (FID) assay for establishing protein DNA binding affinity and selectivity is demonstrated with the examination of the LEF-1 HMG domain binding to hairpin deoxyoligonucleotides containing its commonly accepted consensus sequence 5'-CTTTGWW (W=A or T) and those modified (5'-CTNTGWW) to examine sequences implicated in early studies (5'-CTNTG). The effectiveness of the FID assay coupled with its technically non-demanding experimental use makes it an attractive alternative or complement to selection screening, footprinting or affinity cleavage, and electrophoretic mobility shift assays for detecting, characterizing, and quantitating protein DNA binding affinity and selectivity.     

DNA binding fluorescent proteins for the direct visualization of large DNA molecules

Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely ‘paints’ entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (K_d = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells.     

DNA binding by analogues of the bifunctional intercalator TANDEM

We have used DNase I footprinting to study the binding strength and DNA sequence selectivity of novel derivatives of the quinoxaline bis-intercalator TANDEM. Replacing the valine residues in the cyclic octadepsipeptide with lysines does not affect the selectivity for TpA but leads to a 50-fold increase in affinity. In contrast, replacing both of the quinoxaline chromophores with naphthalene rings abolishes binding, while changing a single ring decreases the affinity, and footprints are observed at only the best binding sites (especially TATATA). By using fragments with different lengths of [(AT) n ], we demonstrate that these ligands bind best to the center of the longer (AT) n tracts.     

Efficient high-throughput discovery of large peptidic hormones and biomarkers

A novel approach is presented for the simultaneous identification and relative quantification of secreted peptides, particularly those that have been historically difficult to analyze in a concerted manner. Peptides exceeding 60 residues with various degrees of post-translational modification were identified on a liquid chromatographic time scale. The approach demonstrates high efficiency pattern-based recognition analysis of complex neuroendocrine peptide sets and enables rapid identification of biomarkers from biological material.     

Establishment of a high-throughput detection system for DNA demethylating agents

Epigenetic alterations underlie various human disorders, including cancer, and this has resulted in the development of drugs targeting epigenetic alterations. Although DNA demethylating agents are one of the major epigenetic drugs, only two compounds—5-azacytidine (5-aza-CR, azacitidine) and 5-aza-2'-deoxycytidine (5-aza-dC, decitabine)—have obtained clinical approval. Here, we aimed to establish a detection system for DNA demethylating agents suitable for a high-throughput screening (HTS) in mammalian cells. We inserted luciferase and EGFP reporter genes under the UCHL1 promoter, which is methylation-silenced in human colon cancers and can be readily demethylated to drive strong expression. Methylated UCHL1 promoter was introduced into HCT116 colon cancer cells, and transfectants with methylated exogenous UCHL1 promoter were obtained. By screening subclones from each of the epigenetically heterogeneous transfectant clones, we finally obtained three optimal subclones that expressed luciferase and EGFP after 5-aza-dC treatment with high signal-to-noise ratios. Nucleosomes with H3K9me2 were present around the exogenous UCHL1 promoter in all three subclones. Using one of the subclones (HML58-3), HTS was conducted using 19,840 small molecules. Two hit compounds were obtained, and these turned out to be 5-aza-dC and 5-aza-CR. The assay system constructed here demonstrates a robust response to DNA demethylating agents, along with high specificity, and will be useful for screening and biological assays in epigenetics.     

PASTE: a high-throughput method for large DNA insertions

Prime editing (PE) enables precise genome editing at targeted locus without inducing double-stranded breaks (DSBs). Despite its precision, PE lacks the tendency to integrate large DNA fragments into the genome. Recently, Yarnall et al. reported clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 and an integrase-based system that conducts targeted integration of large DNA sequences (~36 kb) into the genome more efficiently.     

Determination of binding affinities of triplex forming oligonucleotides using a fluorescent intercalator displacement (FID) assay

The binding affinities of several triplex forming oligonucleotides were determined using a fluorescent intercalator displacement (FID) assay.     

Thiazole orange as the fluorescent intercalator in a high resolution fid assay for determining DNA binding affinity and sequence selectivity of small molecules.

The viability of using thiazole orange as an alternative to ethidium bromide in a fluorescent intercalator displacement (FID) assay is explored by profiling the DNA binding affinity and sequence selectivity of netropsin. Utilizing a library of hairpin deoxyoligonucleotides containing all possible four base-pair sequences, the method provides a high resolution profile of the DNA binding properties of small molecules in a high throughput format.     

DNA binding affinity of pentapeptides selected from combinatorial library.

The combinatorial method has been applied to determine peptide ligands to the duplex DNA by using the solid-state pentapeptide library and the target-DNA conjugated magnetic beads. Seventy-one sequences were determined as ligands for AT duplex. Interestingly, hydrophobic amino acids such as Phe, Ile and Gly were most frequently determined. Relative binding affinity of the selected pentapeptides with the various DNA sequences was estimated by ethidium displacement assay in 10 mM SHE buffer. FQGII constituted of amino acids that were most frequently determined in the random screening showed highest binding affinity to the duplex DNA.     

Focused library development of 2-phenylacrylamides as broad spectrum cytotoxic agents

With our lead compound (E)-3-(4-chlorophenyl)-2-(1H-pyrrole-2-carbonyl)acrylonitrile (1) inducing 50% growth inhibition of 11 cancer cell lines at 27–61 μM, potency enhancements were rapidly established through the synthesis of a series of focused compound libraries. Six highly focused libraries (46 compounds in total) were synthesised. Each library allowed the identification of a new lead compound, viz Library A identified (E)-3-(pentafluorophenyl)-2-(1H-pyrrole-2-carbonyl)acrylonitrile (11) and (E)-3-(1H-indol-3-yl)-2-(1H-pyrrole-2-carbonyl)acrylonitrile (13) as inhibitors with improved cytotoxicity. Synthesis of discrete libraries of amidoacrylamide analogues (Ar–CC(CN)–Ar?Ar–CC(CN)–C(O)NH)–Ar) resulted in a series of analogues significantly more potent that the lead, 1. Three furan three analogues: (E)-3-(5-chlorofuran-2-yl)-2-cyano-N-(4-methoxybenzyl)acrylamide (33), (E)-3-(5-bromofuran-2-yl)-2-cyano-N-(4-methoxybenzyl)acrylamide (34) and (E)-2-cyano-3-(furan-3-yl)-N-(4-methoxybenzyl)acrylamide (37) returned broad spectrum growth inhibition (GI₅₀ values of 5–16 μM). Replacement of the furan moiety with simple aromatics gave an additional three analogues: (E)-2-cyano-N-(4-methoxybenzyl)-3-phenylacrylamide (39), (E)-3-(4-chlorophenyl)-2-cyano-N-(4-methoxybenzyl)acrylamide (41) and (E)-2-cyano-N-(4-methoxyphenyl)-3-(naphthalen-1-yl)acrylamide (45) with GI₅₀ values of 7–24 μM. The final library retained the aromatic substituents but introduced a 3,4-dichlorbenzylamine moiety to afford the 1-naphthyl substituted 52, which was the most potent broad spectrum cytotoxic analogue produced here in with an average GI₅₀ = 8.6 μM. This represents a fivefold potency enhancement relative to 1 and a new cytotoxic scaffold suitable for further development.     

Construction and characterisation of a large DNA insert library from the D genome of wheat

 A large DNA fragment library consisting of 144 000 clones with an average insert size of 119 kb was constructed from nuclear DNA isolated from root and leaf tissue from Triticum tauschii (syn. Aegilops tauschii), the D-genome progenitor of wheat. The library was made in a binary vector that had previously been shown to stably maintain large inserts of foreign DNA in Escherichia coli. The use of root nuclei reduced considerably the proportion of the library containing clones derived from chloroplast DNA. Several experimental parameters were investigated and optimised, leading to a high cloning efficiency. Only three ligations were needed to construct the library which was estimated to be equivalent to 3.7 haploid genomes. The accuracy of this estimation was demonstrated by screening this library with three well-defined probes. One probe containing a glutenin gene sequence identified 5 clones covering at least 230 kb of the Glu-D1 locus and contained the two tightly linked high-molecular-weight glutenin genes Glu-D1x and -D1y. Each of the other two single-copy probes derived from the Cre3 cereal cyst nematode resistance gene locus hybridised with 4 clones containing gene sequences encoding nucleotide binding sites and a leucine-rich region. This is the first representative large-insert DNA library for wheat, and the results indicated that large molecules of wheat DNA can be efficiently cloned, stably maintained and manipulated in a bacterial system. Received: 28 August 1998 / Accepted: 28 November 1998     

The effect of intercalator structure on binding strength and base-pair specificity in DNA interactions

The interaction of naphthothiophene, phenanthrene and anthracene ring systems, which have amide and ester side chains with cationic groups (synthesized from the aromatic acid chlorides and appropriate amines and alcohols), with calf thymus DNA has been investigated by using viscometric titrations, spectrophotometric binding experiments and 1H-, 31P- and 17O-NMR methods. The viscosity and NMR experiments suggest that all of these compounds bind to DNA by intercalation. These experiments and spectrophotometric binding studies, however, indicate that there is considerable variation in the interaction of these compounds with DNA. These variations can all be explained by the geometry of the ring systems, the position of protons adjacent to the side chains, and the relative sizes of the amide and ester side chains. With the naphthothiophene ester and amide, for example, the planar amide cannot rotate into the plane of the naphthothiophene ring whereas the smaller planar ester can. With this ring system the ester has a significantly higher binding constant than the amide derivative. Additional binding studies with poly[d(A-T)2] and poly[d(G-C)2] have shown that all of these compounds bind more strongly to the A-T- than the G-C-containing polymer. Since the ester compounds do not have hydrogen bond donating groups proximate to the aromatic ring, these results suggest a model for the A-T specificity of these compounds that involves a solvent-mediated hydrogen bond between the C-2 carbonyl of thymine and the carbonyl group of the intercalators.     

Evaluation of Mongolian compound library for potential antimalarial and anti-Toxoplasma agents

179 compounds in a Mongolian compound library were investigated for their inhibitory effect on the in vitro growth of Plasmodium falciparum and Toxoplasma gondii. Among these compounds, brachangobinan A at a half-maximal inhibition concentration (IC₅₀) of 2.62 μM and a selectivity index (SI) of 27.91; 2-(2′-hydroxy-5′-O-methylphenyl)-5-(2″,5″-dihydroxyphenyl)oxazole (IC₅₀ 3.58 μM and SI 24.66); chrysosplenetin (IC₅₀ 3.78 μM and SI 15.26); 4,11-di-O-galloylbergenin (IC₅₀ 3.87 μM and SI 13.38); and 2-(2′,5′-dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole (IC₅₀ 6.94 μM and SI 11.48) were identified as potential inhibitors of P. falciparum multiplication. Additionally, tricin (IC₅₀ 12.94 μM and SI > 23.40) was identified as a potential inhibitor of T. gondii multiplication. Our findings represent a good starting point for developing novel antimalarial and anti-Toxoplasma therapeutics from Mongolian compounds.     

Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.     

Function-based isolation of novel enzymes from a large library

Here we describe a high-throughput, quantitative method for the isolation of enzymes with novel substrate specificities from large libraries of protein variants. Protein variants are displayed on the surface of microorganisms and incubated with a synthetic substrate consisting of (1) a fluorescent dye (2) a positively charged moiety (3) the target scissile bond, and (4) a fluorescence resonance energy transfer (FRET) quenching partner. Enzymatic cleavage of the scissile bond results in release of the FRET quenching partner while the fluorescent product is retained on the cell surface, allowing isolation of catalytically active clones by fluorescence-activated cell sorting (FACS). Using a synthetic substrate with these characteristics, we enriched Escherichia coli expressing the serine protease OmpT from cells expressing an inactive OmpT variant by over 5,000-fold in a single round. Screening a library of 6 x 10(5) random OmpT variants by FACS using a FRET peptide substrate with a nonpreferred Arg-Val cleavage sequence resulted in the isolation of variant proteases with catalytic activities enhanced by as much as 60-fold. This approach represents a potentially widely applicable method for high-throughput screening of large libraries on the basis of catalytic turnover.     

Mammalian DNA repair--use of mutants hypersensitive to cytotoxic agents

Mutant mammalian cells hypersensitive to DNA damaging agents are a useful tool for the characterization of DNA repair pathways, and can be used to isolate, by transfection, DNA repair genes and genes that confer basal resistance to cytotoxic agents. These genes may be important in mutagenesis, carcinogenesis and resistance of cancer cells to therapy.     

Assessment of solution-phase positional scanning libraries based on distamycin A for the discovery of new DNA binding agents

The solution-phase synthesis of two 1000-membered positional scanning libraries of distamycin A analogues is described enlisting acid/base liquid-liquid extractions for isolation and purification of all intermediates and final products. The results of their screening for functional activity (L1210 cytotoxic potency) and DNA binding affinity were compared with those derived from libraries containing the same compound members but prepared in a smaller 10-compound mixture format. The positional scanning libraries, which are substantially less demanding to prepare, allowed the accurate detection of the global observations and the clearly more potent activities, but more subtle discoveries and less distinguishable activities were not detected. This is a natural consequence of testing the larger 100-compound mixtures and the relative insensitivity of the assays to the contribution of any single, uniquely acting compound in the mixture. Thus, the disadvantages associated with the loss of some information contained within the library must be balanced against the advantages of the ease of library synthesis and judged in light of the library screening objectives.     

Analysis of large gene databases for discovery of novel therapeutic agents

During the 1980s and early 1990s the recombinant DNA revolution provided a vital source of therapeutic targets and agents for pharmaceutical research. However, during the early 1990s, it became apparent that the identification and cloning of novel human cDNAs was a rate limiting step in drug discovery and that new technological approaches were required to address the challenge. There was an increasing realisation that the new science of 'genomics', together with the associated large gene sequence databases, would provide a radically new means of generating targets. SmithKline Beecham has been at the forefront of this breakthrough in pharmaceutical research. The productivity of this strategy is illustrated by reference to our work on novel enzymes, chemokines and receptors and new approaches linking genes to pathological processes.