Improved Antiviral Activity of a Polyamide Against High-Risk Human Papillomavirus Via N-Terminal Guanidinium Substitution.

We report the synthesis of two novel pyrrole-imidazole polyamides with N-terminal guanidinium or tetramethylguanidinium groups and evaluate their antiviral activity against three cancer-causing human papillomavirus strains. Introduction of guanidinium improves antiviral activity when compared to an unsubstituted analog, especially in IC90 values. These substitutions change DNA-binding preferences, while binding affinity remains unchanged.

Enhancing sequence-specific cleavage of RNA within a duplex region: incorporation of 1,3-propanediol linkers into oligonucleotide conjugates of serinol-terpyridine.

The syntheses and RNA cleavage efficiencies of a new series of oligonucleotide conjugates of Cu(II)-serinol-terpyridine and 1,3-propanediol are reported. These reagents, termed ribozyme mimics, were designed such that they would yield multiple unpaired RNA residues directly opposite the site of the RNA cleavage catalyst upon ribozyme mimic-RNA duplex formation. This design effect was implemented using the 1,3-propanediol linker 3, which mimics the three-carbon spacing between the 5'- and 3'-hydroxyls of a natural nucleotide. Incorporation of one or more of these 1,3-propanediol linkers at positions directly adjacent to the serinol-terpyridine modification in the ribozyme mimic DNA strand resulted in cleavage at multiple phosphates in a complementary 31-mer RNA target sequence. The linkers effectively created artificial mismatches in the RNA-DNA duplexes, rendering the opposing RNA residues much more susceptible to cleavage via the transesterification/hydrolysis pathway. The RNA cleavage products produced by the various mimics correlated directly with the number and locations of the linkers in their DNA strands, and the most active ribozyme mimic in the series exhibited multiple turnover in the presence of excess 31-mer RNA target.

Efficient new ribozyme mimics: direct mapping of molecular design principles from small molecules to macromolecular, biomimetic catalysts.

Dramatic improvements in ribozyme mimics have been achieved by employing the principles of small molecule catalysis to the design of macromolecular, biomimetic reagents. Ribozyme mimics derived from the ligand 2,9-dimethylphenanthroline (neocuproine) show at least 30-fold improvements in efficiency at sequence-specific RNA cleavage when compared with analogous o-phenanthroline- and terpyridine-derived reagents. The suppression of hydroxide-bridged dimers and the greater activation of coordinated water by Cu(II) neocuproine (compared with the o-phenanthroline and terpyridine complexes) better allow Cu(II) to reach its catalytic potential as a biomimetic RNA cleavage agent. This work demonstrates the direct mapping of molecular design principles from small-molecule cleavage to macromolecular cleavage events, generating enhanced biomimetic, sequence-specific RNA cleavage agents.

Hydrolysis of phosphates, esters and related substrates by models of biological catalysts.

Why study hydrolases, and why model them? First, hydrolases themselves are of fundamental importance and utility. Examples of their utility in organic synthesis include kinetic resolutions of optical isomers. Restriction endonucleases (DNA hydrolases) are key tools for biotechnology and are vital biological catalysts. Peptidases are necessary for protein digestion and can be harnessed to perform the reverse reaction (peptide synthesis). Thus, for these and many other reasons, hydrolases receive the attention of fundamental and applied research. Models of hydrolases can contribute to our understanding of reaction mechanisms and may also supplant the enzymes as useful catalysts under some conditions. Altering or even increasing the specificity of natural catalysts are also goals of these model studies.

The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.

Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.

Modulation of RNase H activity by modified DNA probes: major groove vs minor groove effects.

We have previously prepared ribozyme mimics and chemical nucleases from modified DNA containing pendant bipyridine and terpyridine groups. The ability of these modified DNA probes to support RNase H cleavage of complementary RNA is described. DNA/RNA duplexes were formed using DNA probes designed to deliver metal complexes via either the major groove or the minor groove of the duplex. The duplexes were treated with Escherichia coli RNase H. Modifications in the major groove produced the same RNA cleavage pattern as unmodified DNA probes. However, minor groove substituents inhibited RNA cleavage over a four-base region. Comparison was made with a DNA probe containing a 2'-OMe modification. Our results support enzyme binding in the minor groove of a DNA/RNA duplex. We do not observe cleavage directly across from the modified nucleoside. The RNA cleavage efficiency effected by RNase H and a DNA probe decreases as follows: unmodified DNA > or = C-5 modified DNA >> c2'-modified DNA > C1'-modified DNA. Results with 28-mer RNA substrates roughly parallel those obtained with a 159-mer RNA target. The differences observed between low and high MW RNA substrates can be explained by a much higher enzyme-substrate binding constant for the high MW target.

DNA enzymes: new-found chemical reactivity.

DNA is generally considered to be chemically rather inert, but self-cleaving DNA molecules have recently been isolated from in vitro selection experiments; their properties advance the idea that, like RNA, DNA can serve as both a catalyst and an information storage medium.

Short tandem repeat typing by capillary array electrophoresis: comparison of sizing accuracy and precision using different buffer systems.

Polymorphic microsatellite markers are widely used in gene discovery and mapping, human identification, agricultural genetics, and diagnosis of triplet-repeat expansion disorders. Reliable genotyping of these markers requires polymerase chain reaction (PCR) amplification and very-high-resolution electrophoresis. Capillary array electrophoresis offers extremely fast, high-resolution separation of DNA and more automated sample processing because labor-intensive slab-gel pouring and sample loading are eliminated. We report a simple, reliable procedure for preparing PCR samples for electrokinetic injection into capillaries using a 96-well tray and float dialysis. We developed an improved sizing standard for genotyping and used it to evaluate systematically the sizing accuracy and precision of low-viscosity, replaceable matrix formulations. Our study sizing over 28,000 alleles yielded an average precision of +/- 0.12 bp for fragments up to 350 bp. Low-viscosity formulations permit low-pressure matrix injection (40 psi) and a turnaround time of 70 min for 48-96 samples.

High-throughput DNA sequencing on a capillary array electrophoresis system.

A capillary array electrophoresis apparatus capable of running and analyzing 48 DNA sequencing samples simultaneously has been constructed. The instrument uses a replaceable sieving buffer and incorporates a convenient method for introducing the buffer into the capillaries. Data from laser-induced fluorescence are collected as four separate images, one for each optical channel. The integrated data analysis software employs an open architecture that allows use of any DNA base-calling algorithm. DNA sequencing runs are completed in approx. 1 hr (approximately 500 bases), and instrument turnaround time between runs is less than 15 min. Overall, the instrument throughput is on the order of 720 templates/day, or 360,000 bases/day.

Quantitative analysis of electrophoresis data: novel curve fitting methodology and its application to the determination of a protein-DNA binding constant.

A computer program, GelExplorer, which uses a new methodology for obtaining quantitative information about electrophoresis has been developed. It provides a straightforward, easy-to-use graphical interface, and includes a number of features which offer significant advantages over existing methods for quantitative gel analysis. The method uses curve fitting with a nonlinear least-squares optimization to deconvolute overlapping bands. Unlike most curve fitting approaches, the data is treated in two dimensions, fitting all the data across the entire width of the lane. This allows for accurate determination of the intensities of individual, overlapping bands, and in particular allows imperfectly shaped bands to be accurately modeled. Experiments described in this paper demonstrate empirically that the Lorentzian lineshape reproduces the contours of an individual gel band and provides a better model than the Gaussian function for curve fitting of electrophoresis bands. Results from several fitting applications are presented and a discussion of the sources and magnitudes of uncertainties in the results is included. Finally, the method is applied to the quantitative analysis of a hydroxyl radical footprint titration experiment to obtain the free energy of binding of the lambda repressor protein to the OR1 operator DNA sequence.

Implementation of a capillary array electrophoresis instrument.

A capillary array electrophoresis (CAE) apparatus capable of running and analyzing DNA samples in 48 capillaries simultaneously has been constructed. The capillaries are individually replaceable, and sieving buffer can be easily pumped in and out of the capillary array as necessary. Samples are injected electrokinetically from polymerase chain reaction (PCR, Hoffmann-LaRoche, Nutley, NJ, U.S.A.) tubes arranged in a 6 x 8 format and are detected by laser-induced fluorescence. Data analysis software has been developed for semiautomatic analysis, including peak finding and DNA fragment sizing. The system represents a robust apparatus for the rapid and convenient analysis of DNA fragments in a high-throughout environment.

DNA sequencing by capillary electrophoresis with a hydroxyethylcellulose sieving buffer.

Capillary electrophoresis (CE) is capable of rapid, high-resolution separations of DNA. The technique has been adopted for both ssDNA and dsDNA applications. In order to make CE more convenient and cost-effective, replaceable sieving buffers have recently been developed. For DNA sequencing, the most successful of these replaceable buffers have been carefully polymerized and purified viscous linear polyacrylamide solutions. However, the hazards of acrylamide are well documented, and the care required to prepare the appropriate molecular weight polymer make this approach less than ideal. We report use of a replaceable sieving buffer suitable for DNA sequencing by CE that is easy to prepare and uses commercially available, non-toxic hydroxyethylcellulose.

Ribozyme mimics as catalytic antisense reagents.

Viral and fungal infections and some cancers may be described as diseases that are characterized by the expression of certain unwanted proteins. They could be termed induced genetic disorders, with induction provided by mutation or infection. A comprehensive method to inactivate injurious genes based on their nucleic acid sequences has the potential to provide effective antiviral and anticancer agents with greatly reduced side effects. We describe a chemical approach to such gene-specific pharmaceutical agents. Our initial efforts have been to develop new chemical reagents that can carry out catalytic destruction of specific mRNA sequences. We chose hydrolysis as a chemical means of destruction, because hydrolysis is compatible with living cells. Our sequence-specific catalytic RNA hydrolysis reagents may be described as functional ribozyme mimics. Reactivity is provided by small-molecule catalysts, such as metal complexes. Specificity is provided by oligonucleotide probes. Here we report initial results on the sequence-specific, hydrolytic cleavage of mRNA from the HIV gag gene, using a ribozyme mimic. The reagent is composed of a terpyridylCu(II) complex for cleavage activity and an oligonucleotide for sequence specificity.

A new trinuclear complex of platinum and iron efficiently promotes cleavage of plasmid DNA.

The compound [[Pt(trpy)]2Arg-EDTA]+ is synthesized in five steps, purified, and characterized by 1H, 13C, and 195Pt NMR spectroscopy, mass spectrometry, UV-vis spectrophotometry, and elemental analysis. The binuclear [[(Pt(trpy)]2Arg]3+ moiety binds to double-stranded DNA, and the chelating EDTA moiety holds metal cations. In the presence of ferrous ions and the reductant dithiothreitol, the new compound cleaves DNA. It cleaves a single strand in the pBR322 plasmid nearly as efficiently as methidiumrpropyl-EDTA (MPE), and it cleaves a restriction fragment of the XP10 plasmid nonselectively and more efficiently than [Fe(EDTA)]2-. The mechanism of cleavage was studied in control experiments involving different transition-metal ions, superoxide dismutase, catalase, glucose oxidase with glucose, metal-sequestering agents, and deaeration. These experiments indicate that adventitious iron and copper ions, superoxide anion, and hydrogen peroxide are not involved and that dioxygen is required. The cleavage apparently is done by hydroxyl radicals generated in the vicinity of the DNA molecule. The reagent [[Pt(trypy)]2Arg-EDTA]+ differs from methidiumpropyl-EDTA in not containing an intercalator. This difference in binding modes between the binuclear platinum(II) complex and the planar heterocycle may cause useful differences between the two reagents in cleavage of nucleic acids.

Structure of DNA in a nucleosome core at high salt concentration and at high temperature.

We have used hydroxyl radical cleavage of DNA to probe the organization of the nucleosome core at high salt concentration and high temperature. The rotational and translational positioning of a DNA fragment, containing part of the Xenopus borealis 5S RNA gene, on the histone octamer is maintained between salt concentrations of 0.0 and 0.8 M NaCl and between temperatures of 0 and 75 degrees C. These results provide evidence that the energy of bending DNA around the nucleosome is independent of salt concentration and temperature in this range. They illustrate the severe energetic requirements necessary to displace DNA from previously organized contacts with histones in the nucleosome core.

The histone core exerts a dominant constraint on the structure of DNA in a nucleosome.

We have examined the structures of unique sequence, A/T-rich DNAs that are predicted to be relatively rigid [oligo(dA).oligo(dT)], flexible [oligo[d(A-T)]], and curved, using the hydroxyl radical as a cleavage reagent. A 50-base-pair segment containing each of these distinct DNA sequences was placed adjacent to the T7 RNA polymerase promoter, a sequence that will strongly position nucleosomes. The final length of the DNA fragments was 142 bp, enough DNA to assemble a single nucleosome. Cleavage of DNA in solution, while bound to a calcium phosphate crystal, and after incorporation into a nucleosome is examined. We find that the distinct A/T-rich DNAs have very different structural features in solution and helical periodicities when bound to a calcium phosphate. In contrast, the organization of the different DNA sequences when associated with a histone octamer is very similar. We conclude that the histone core exerts a dominant constraint on the structure of DNA in a nucleosome and that inclusion of these various unique sequences has only a very small effect on overall nucleosome stability and structure.