Sequence specific alkylation of DNA by hairpin pyrrole-imidazole polyamide conjugates

BACKGROUND: Pyrrole-imidazole polyamides are synthetic ligands that recognize predetermined sequences in the minor groove of DNA with affinities and specificities comparable to those of DNA-binding proteins. As a result of their DNA-binding properties, polyamides could deliver reactive moieties for covalent reaction at specific DNA sequences and thereby inhibit DNA-protein interactions. Site-specific alkylation of DNA could be a useful tool for regulating gene expression. As a minimal first step, we set out to design and synthesize a class of hairpin polyamides equipped with DNA alkylating agents and characterize the specificity and yield of covalent modification. RESULTS: Bis(dichloroethylamino)benzene derivatives of the well-characterized chlorambucil (CHL) were attached to the gamma turn of an eight-ring hairpin polyamide targeted to the HIV-1 promoter. We found that a hairpin polyamide-CHL conjugate binds and selectively alkylates predetermined sites in the HIV promoter at subnanomolar concentrations. Cleavage sites were determined on both strands of a restriction fragment containing the HIV-1 promoter, revealing good specificity and a high yield of alkylation. CONCLUSIONS: The ability of polyamide-CHL conjugates to sequence specifically alkylate double-stranded DNA in high yield and at low concentrations sets the stage for testing their use as regulators of gene expression in cell culture and ultimately in complex organisms.     

Simultaneous AFM manipulation and fluorescence imaging of single DNA strands.

We report combined atomic force and far-field fluorescence microscopic experiments which allow the simultaneous atomic force manipulation and optical observation of individual dye-labeled DNA molecules. A detailed understanding of the binding properties of DNA to different transparent surfaces is prerequisite for these investigations. Atomic force spectroscopy and fluorescence microscopy of single DNA strands yielded detailed insight into two different types of DNA binding onto transparent polylysine-coated and silanized glass surfaces. We subsequently demonstrate how the different binding can be exploited to perform two types of nanomanipulation experiments: On polylysine, strong electrostatic interactions over the whole length of the DNA strand enable the writing of micrometer-sized patterns. By contrast, the strong pointwise attachment of DNA to silanized surfaces allows horizontal stretching of single DNA strands to lengths exceeding 1.6 times the contour length of the DNA strand. With this new approach it is possible to directly observe the rupture of the strongly bonded DNA strand.     

Silver nanoparticle-DNA bionanoconjugates bearing a discrete number of DNA ligands

Silver nanoparticles (AgNPs), which are stable in strongly ionic solutions and appear as a single sharp band during gel electrophoresis, are synthesized by a facile one-pot process, allowing for the first time realization of AgNP-DNA bio-nano-conjugates bearing a discrete number of DNA ligands.     

Reaction kinetics study of short DNA strands using a maximum entropy method and nonlinear curve fitting

It is important to understand the formation of double-strand DNA (dsDNA) in a salt solution because it is one of the key reactions in life. A short cDNA strand pair was designed, and each single-strand DNA (ssDNA) was attached to a fluorescent dye that was either a donor or an acceptor of fluorescence resonance energy transfer. The fluorescence intensity was expected to change as time passed as the complementary pairs of ssDNAs formed dsDNAs. The concentration of dsDNA was theoretically calculated, and the measured data were consistent with theoretical results. The analysis of the nonlinear fitting method and the maximum entropy method detected that the reaction curve contains two major types of kinetics that likely represent the formation of dsDNA and mismatching.     

Circular dichroism study of enzymatic manipulation on magnetic and metallic DNA template nanowires

Circular dichroism spectroscopy (CD) was used to examine the mechanism of endonuclease clipping and ligation of the DNA template nanowires. The biomolecular manipulation of the DNA template is compared for both metallic (Au) and magnetic (Fe₂O₃ and CoFe₂O₄) nanowires. The dependence of nanoparticle (NP) concentration on enzymatic clipping and DNA ligation was studied, in addition to performing absorbance and thermal melting experiments. Low-NP concentration preserved and digested the DNA template structure. Yet, at higher NP concentrations, the DNA template began to denature before enzyme addition. It was also observed that ligation of the digested DNA occurred more efficiently at low-NP concentrations. These results provide significant information on structural alteration and biorecognition effectiveness of the DNA template after enzymatic manipulation.     

Circular dichroism study of enzymatic manipulation on magnetic and metallic DNA template nanowires

Circular dichroism spectroscopy (CD) was used to examine the mechanism of endonuclease clipping and ligation of the DNA template nanowires. The biomolecular manipulation of the DNA template is compared for both metallic (Au) and magnetic (Fe₂O₃ and CoFe₂O₄) nanowires. The dependence of nanoparticle (NP) concentration on enzymatic clipping and DNA ligation was studied, in addition to performing absorbance and thermal melting experiments. Low-NP concentration preserved and digested the DNA template structure. Yet, at higher NP concentrations, the DNA template began to denature before enzyme addition. It was also observed that ligation of the digested DNA occurred more efficiently at low-NP concentrations. These results provide significant information on structural alteration and biorecognition effectiveness of the DNA template after enzymatic manipulation.     

Site-specific oxidative cleavage of DNA by metallosalen-DNA conjugates

Ni-salen-DNA conjugates, prepared by template-directed synthesis, targeted oxidative adduct formation and strand scission at deoxyguanosine sites in complementary DNA strands of Watson-Crick duplexes.     

DNA manipulation with elastomeric nanostructures fabricated by soft-moulding of a FIB-patterned stamp

A Focused Ion Beam (FIB)-patterned silicon mould is used to fabricate elastomeric nanostructures, whose cross-section can be dynamically and reversibly tuned by applying a controlled mechanical stress. Direct-write, based on FIB milling, allows the fabrication of nanostructures with a variety of different geometries, aspect ratio, spacing and distribution offering a higher flexibility compared to other nanopatterning approaches. Moreover, a simple double replication process based on poly(dimethylsiloxane) permits a strong reduction of the fabrication costs that makes this approach well-suited for the production of low cost nanofluidic devices. DNA stretching and single molecule manipulation capabilities of these platforms have been successfully demonstrated.     

Sequence-specific fluorescence detection of DNA by polyamide-thiazole orange conjugates

Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation.     

Site-selective RNA cleavage by DNA bearing a base pair-mimic nucleoside

We have synthesized the deoxyadenosine derivative tethering a phenyl group (X), which mimics the Watson-Crick A/T base pair. The RNA/DNA hybrid duplexes containing X in the middle of the DNA sequence showed a similar thermal stability regardless of the ribonucleotide species (A, G, C, or U) opposite to X, probably because of the phenyl group stacking inside of the duplex accompanied by the opposite ribonucleotide base flipped in an extrahelical position. The RNA strand hybridized with the DNA strand bearing X was cleaved on the 3'-side of the ribonucleotide opposite to X in the presence of MgCl2, and the RNA sequence to be cleaved was not restricted. The site-specific RNA hydrolysis suggests that the DNA strand bearing X has the advantage of the site-selective base flipping in the target sequence and the development of a "universal deoxyribozyme" to exclusively cleave a target RNA sequence.     

Metal-complex/DNA conjugates: a versatile building block for DNA nanoarrays

The use of DNA networks as templates for forming nanoarrays of metallic centres shows an exciting potential to generate addressable nanostructures. Inorganic units can be photoactive, electroactive and/or can possess magnetic and catalytic properties and can adopt different spatial arrangements due to their varied coordination nature. All these properties influence both the structure and function of passive DNA scaffolds and provide DNA nanostructures as a new platform for new materials in emerging technologies, such as nanotechnology, biosensing or biocomputing.     

Sequence-specific trapping of topoisomerase I by DNA binding polyamide-camptothecin conjugates

Hairpin pyrrole-imidazole polyamides are synthetic ligands that bind in the minor groove of DNA with affinities and specificities comparable to those of DNA binding proteins. Three polyamide-camptothecin conjugates 1-3 with linkers varying in length between 7, 13, and 18 atoms were synthesized to trap the enzyme Topoisomerase I and induce cleavage at predetermined DNA sites. One of these, polyamide-camptothecin conjugate 3 at nanomolar concentration (50 nM) in the presence of Topo I (37 degrees C), induces DNA cleavage between three and four base pairs from the polyamide binding site in high yield (77%).     

Double-stranded DNA dissociates into single strands when dragged into a poor solvent

DNA displays a richness of biologically relevant supramolecular structures, which depend on both sequence and ambient conditions. The effect of dragging double-stranded DNA (dsDNA) from water into poor solvent on the double-stranded structure is still unclear because of condensation. Here, we employed single molecule techniques based on atomic force microscopy and molecular dynamics (MD) simulations to investigate the change in structure and mechanics of DNA during the ambient change. We found that the two strands are split apart when the dsDNA is pulled at one strand from water into a poor solvent. The findings were corroborated by MD simulations where dsDNA was dragged from water into poor solvent, revealing details of the strand separation at the water/poor solvent interface. Because the structure of DNA is of high polarity, all poor solvents show a relatively low polarity. We speculate that the principle of spontaneous unwinding/splitting of dsDNA by providing a low-polarity (in other word, hydrophobic) micro-environment is exploited as one of the catalysis mechanisms of helicases.     

Thermodynamic properties of branched DNA complexes with full-matched and mismatched DNA strands

Complexes consisting of a branched DNA with full-matched and mismatched DNA strands were prepared, and the cross-linking of the DNA strands and their diastereochemistry affected the stability of the complexes and the thermodynamics of the complex formation.     

Assembly of highly aligned DNA strands onto Si chips

This paper reports a robust and efficient approach to assemble highly aligned DNA strands onto Si chips. The method combines advantages from molecular combing and microcontact printing to realize controlling both the density and direction of DNA strands on the Si chip. In addition, it also can be utilized to prepare stretched DNA structures on solid surfaces. Compared to approaches that use molecular combing directly on silanated surfaces, the stretched single-chain DNA structures are straighter. Furthermore, by exploiting the hydrophobic property of the intrinsic poly(dimethylsiloxane) stamp, this study also describes a simple way to produce straight bundled DNA arrays on Si and other substrates.     

Purification of genomic DNA by short monolithic columns

The isolation and purification of nucleic acids is essential for many procedures in molecular biology. After showing that bacterial and eukaryotic genomic DNA can be specifically bound to the CIM DEAE monolithic column, this characteristic was exploited in development of a simple and fast chromatographic procedure for isolation and purification of genomic DNA from cell lysates that does not include the usage of toxic organic solutions. The purity and the quality of the isolate as well as the duration of the procedure was similar to other chromatographic methods used today for isolation of genomic DNA, but the initial sample volume was not restricted.