Megan E. Núñez

Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretching

We used single DNA molecule stretching to investigate DNA intercalation by ethidium and three ruthenium complexes. By measuring ligand-induced DNA elongation at different ligand concentrations, we determined the binding constant and site size as a function of force. Both quantities depend strongly on force and, in the limit of zero force, converge to the known bulk solution values, when available. This approach allowed us to distinguish the intercalative mode of ligand binding from other binding modes and allowed characterization of intercalation with binding constants ranging over almost six orders of magnitude, including ligands that do not intercalate under experimentally accessible solution conditions. As ligand concentration increased, the DNA stretching curves saturated at the maximum amount of ligand intercalation. The results showed that the applied force partially relieves normal intercalation constraints. We also characterized the flexibility of intercalator-saturated dsDNA for the first time.

Concentration and separation of hypoglycemic drugs using solid-phase extraction-capillary electrophoresis

Solid-phase extraction-capillary electrophoresis (SPE-CE) is a technique whereby very dilute analytes may be selectively extracted from a sample matrix and concentrated on-line for analysis. This study describes the first phase in the development of a method exploiting this technique for the direct analysis of hypoglycemic drugs in urine. Effective separation and detection of six sulfonylurea drug standards at concentrations below the detection limit of conventional capillary electrophoretic techniques is shown to be attainable. Since surfactant interfered with the on-line concentration process, non-MEKC (micellar electrokinetic chromatography) separation conditions were defined. Using 250 mM borate/5 mM phosphate at pH 8.4, all drugs in a mixture at 285 ng/ml were effectively extracted, concentrated from an injected volume of 2.5 microliters, non-selectively desorbed with an organic-based elution buffer and electrophoretically resolved. Sample loading was found to be linear in the 0.12-1.9 microliters range and drugs in a volume of up to 190 microliters could be concentrated and detected with a sensitivity of approximately 5 ng/ml. Not only was resolution of the desorbed material uncompromised by the presence of the SPE-tip, but separation of glipizide and glyburide was observed despite the fact that these drugs were unresolved under the same separation conditions by standard capillary zone electrophoresis (CZE). From these results, it is clear that SPE-CE not only increases the sensitivity for detection but that selectivity may be altered due to chromatographic processes occurring on the solid-phase resin.

Probing DNA charge transport with metallointercalators.

A wide range of experiments have emerged recently regarding charge transport through DNA, including spectroscopic studies of rates of DNA-mediated electron transfer and biochemical studies of DNA base oxidation over long distances. These experiments have, in turn, led to new proposals about the way in which charge moves through DNA and have prompted the consideration of physiological roles for DNA electron transfer. Importantly, metallointercalators have been key players in many of these experiments. Metallointercalators provide critical probes to examine the migration of charge through the DNA base stack.

Quantitative changes in the elasticity and adhesive properties of Escherichia coli ZK1056 prey cells during predation by bdellovibrio bacteriovorus 109J.

Atomic force microscopy (AFM) was used to explore the changes that occur in Escherichia coli ZK1056 prey cells while they are being consumed by the bacterial predator Bdellovibrio bacteriovorus 109J. Invaded prey cells, called bdelloplasts, undergo substantial chemical and physical changes that can be directly probed by AFM. In this work, we probe the elasticity and adhesive properties of uninvaded prey cells and bdelloplasts in a completely native state in dilute aqueous buffer without chemical fixation. Under these conditions, the rounded bdelloplasts were shown to be shorter than uninvaded prey cells. More interestingly, the extension portions of force curves taken on both kinds of cells clearly demonstrate that bdelloplasts are softer than uninvaded prey cells, reflecting a decrease in bdelloplast elasticity after invasion by Bdellovibrio predators. On average, the spring constant of uninvaded E. coli cells (0.23 +/- 0.02 N/m) was 3 times stiffer than that of the bdelloplast (0.064 +/- 0.001 N/m) when measured in a HEPES-metals buffer. The retraction portions of the force curves indicate that compared to uninvaded E. coli cells bdelloplasts adhere to the AFM tip with much larger pull-off forces but over comparable retraction distances. The strength of these adhesion forces decreases with increasing ionic strength, indicating that there is an electrostatic component to the adhesion events.

Characterizing Pilus-Mediated Adhesion of Biofilm-Forming E. coli to Chemically Diverse Surfaces Using Atomic Force Microscopy

Biofilms are complex communities of microorganisms living together at an interface. Because biofilms are often associated with contamination and infection, it is critical to understand how bacterial cells adhere to surfaces in the early stages of biofilm formation. Even harmless commensal Escherichia coli naturally forms biofilms in the human digestive tract by adhering to epithelial cells, a trait that presents major concerns in the case of pathogenic E. coli strains. The laboratory strain E. coli ZK1056 provides an intriguing model system for pathogenic E. coli strains because it forms biofilms robustly on a wide range of surfaces.E. coli ZK1056 cells spontaneously form living biofilms on polylysine-coated AFM cantilevers, allowing us to measure quantitatively by AFM the adhesion between native biofilm cells and substrates of our choice. We use these biofilm-covered cantilevers to probe E. coli ZK1056 adhesion to five substrates with distinct and well-characterized surface chemistries, including fluorinated, amine-terminated, and PEG-like monolayers, as well as unmodified silicon wafer and mica. Notably, after only 0–10 s of contact time, the biofilms adhere strongly to fluorinated and amine-terminated monolayers as well as to mica and weakly to “antifouling” PEG monolayers, despite the wide variation in hydrophobicity and charge of these substrates. In each case the AFM retraction curves display distinct adhesion profiles in terms of both force and distance, highlighting the cells’ ability to adapt their adhesive properties to disparate surfaces. Specific inhibition of the pilus protein FimH by a nonhydrolyzable mannose analogue leads to diminished adhesion in all cases, demonstrating the critical role of type I pili in adhesion by this strain to surfaces bearing widely different functional groups. The strong and adaptable binding of FimH to diverse surfaces has unexpected implications for the design of antifouling surfaces and antiadhesion therapies.

Hidden in plain sight: subtle effects of the 8-oxoguanine lesion on the structure, dynamics, and thermodynamics of a 15-base pair oligodeoxynucleotide duplex.

The base lesion 8-oxoguanine is formed readily by oxidation of DNA, potentially leading to G → T transversion mutations. Despite the apparent similarity of 8-oxoguanine-cytosine base pairs to normal guanine-cytosine base pairs, cellular base excision repair systems effectively recognize the lesion base. Here we apply several techniques to examine a single 8-oxoguanine lesion at the center of a nonpalindromic 15-mer duplex oligonucleotide in an effort to determine what, if anything, distinguishes an 8-oxoguanine-cytosine (8oxoG-C) base pair from a normal base pair. The lesion duplex is globally almost indistinguishable from the unmodified parent duplex using circular dichroism spectroscopy and ultraviolet melting thermodynamics. The DNA mismatch-detecting photocleavage agent Rh(bpy)(2)chrysi(3+) cleaves only weakly and nonspecifically, revealing that the 8oxoG-C pair is locally stable at the level of the individual base pairs. Nuclear magnetic resonance spectra are also consistent with a well-conserved B-form duplex structure. In the two-dimensional nuclear Overhauser effect spectra, base-sugar and imino-imino cross-peaks are strikingly similar between parent and lesion duplexes. Changes in chemical shift due to the 8oxoG lesion are localized to its complementary cytosine and to the 2-3 bp immediately flanking the lesion on the lesion strand. Residues further removed from the lesion are shown to be unperturbed by its presence. Notably, imino exchange experiments indicate that the 8-oxoguanine-cytosine pair is strong and stable, with an apparent equilibrium constant for opening equal to that of other internal guanine-cytosine base pairs, on the order of 10(-6). This collection of experiments shows that the 8-oxoguanine-cytosine base pair is incredibly stable and similar to the native pair.

Atomic Force Microscopy of Bacterial Communities

This chapter discusses atomic force microscopy (AFM) for the benefit of microbiologists who are interested in using this technique to examine the structures and dynamics of bacteria. AFM is a powerful technique for imaging biological samples at the nanometer to micrometer scale under nondestructive conditions. In order to be imaged with AFM, bacteria must be supported by a surface, which presents challenges because many laboratory strains of bacteria are planktonic. Still, in nature many bacteria live at surfaces and interfaces. This chapter discusses the benefits and difficulties of different methods that have been used to support bacteria on surfaces for AFM imaging and presents two methods in detail used to successfully grow and image bacteria at solid-liquid and solid-air interfaces. Using these methods it is possible to study bacterial morphology and interactions in a native state. These explorations by AFM have important applications to the study of different kinds of bacteria, interfacial bacterial communities, and biofilms.

Damage to DNA by long-range charge transport.

Photochemical reactions on DNA assemblies containing tethered photooxidants, particularly metallointercalating photooxidants, have been critical in establishing that permanent damage to DNA bases can be generated as a result of radical migration from a remote site on the DNA duplex. Induction of a 1-electron deficiency in the oxidant attached covalently to the DNA remote from the oxidizable site leads to this “chemistry at a distance,” caused by efficient charge transport through the DNA base pair stack. Double helical DNA may be unique as a polymeric assembly in solution because of this interior core of stacked aromatic heterocyclic base pairs. Similarly stacked solid-state materials tend to be conducting along the stacking direction. This chapter describes the design and construction of DNA assemblies used to probe long-range oxidative damage in DNA. It also includes methodology for the oxidative repair of a thymine dimer lesion in DNA, as this “chemistry at a distance” also depends on long-range charge transfer through the DNA base pair stack.

Rapid isolation of host-independent Bdellovibrio bacteriovorus

A new method of isolating host-independent Bdellovibrio bacteriovorus has been developed. Filtered suspensions of host-dependent cells are dropped in small volumes onto 0.2 microm membranes laid on rich media agar. Significant growth is observed within 1-2 days; these cells were confirmed to be B. bacteriovorus using microscopic observations and PCR.

Thymine Dimer-Induced Structural Changes to the DNA Duplex Examined with Reactive Probes

Despite significant progress in the past decade, questions still remain about the complete structural, dynamic, and thermodynamic effect of the cis-syn cyclobutane pyrimidine dimer lesion (hereafter called the thymine dimer) on double-stranded genomic DNA. We examined a 19-mer oligodeoxynucleotide duplex containing a thymine dimer lesion using several small, base-selective reactive chemical probes. These molecules probe whether the presence of the dimer causes the base pairs to be more accessible to the solution, either globally or adjacent to the dimer. Though all of the probes confirm that the overall structure of the dimer-containing duplex is conserved compared to that of the undamaged parent duplex, reactions with both diethyl pyrocarbonate and Rh(bpy)(2)(chrysi)(3+) indicate that the duplex is locally destabilized near the lesion. Reactions with potassium permanganate and DEPC hint that the dimer-containing duplex may also be globally more accessible to the solution through a subtle shift in the double-stranded DNA ↔ single-stranded DNA equilibrium. To begin to distinguish between kinetic and thermodynamic effects, we determined the helix melting thermodynamic parameters for the dimer-containing and undamaged parent duplexes by microcalorimetry and UV melting. The presence of the thymine dimer causes this DNA duplex to be slightly less stable enthalpically but slightly less unstable entropically at 298 K, causing the overall free energy of duplex melting to remain unchanged by the dimer lesion within the error of the experiment. Here we consider these results in the context of what has been learned about the thymine dimer lesion from NMR, X-ray crystallographic, and molecular biological methods.