Single-Molecule Imaging Reveals Conformational Manipulation of Holliday Junction DNA by the Junction Processing Protein RuvA

Interactions between DNA and motor proteins regulate nearly all biological functions of DNA such as gene expression, DNA replication and repair, and transcription. During the late stages of homologous recombination (HR), the <i>Escherichia coli</i> recombination machinery, RuvABC, resolves the four-way DNA motifs called Holliday junctions (HJs) that are formed during exchange of nucleotide sequences between two homologous duplex DNA. Although the formation of the RuvA–HJ complex is known to be the first critical step in the RuvABC pathway, the mechanism for the binding interaction between RuvA and HJ has remained elusive. Here, using single-molecule fluorescence resonance energy transfer (smFRET) and ensemble analyses, we show that RuvA stably binds to the HJ, halting its conformational dynamics. Our FRET experiments in different ionic environments created by Mg²⁺ and Na⁺ ions suggest that RuvA binds to the HJ via electrostatic interaction. Further, while recent studies have indicated that the HR process can be modulated for therapeutic applications by selective targeting of the HJ by chemotherapeutic drugs, we investigated the effect of drug-modified HJ on binding. Using cisplatin as a proof-of-concept drug, we show that RuvA binds to the cisplatin-modified HJ as efficiently as to the unmodified HJ, demonstrating that RuvA accommodates for the cisplatin-introduced charges and/or topological changes on the HJ

CD experiments of ILPR-I3 at different pH and temperature in a 10 mM sodium phosphate buffer with 100 mM KCl and 5 µM DNA concentration.

<p>(A) CD spectra of the ILPR-I3 in pH 4.5–8.0. (B) Peak wavelength <i>vs</i> pH for the ILPR-I3 (obtained from (A)) and ILPR-I4 (obtained from the CD spectra of the ILPR-I4 at pH 4.5–8.0, data not shown). (C) CD spectra acquired at 23–68°C (pH 5.5). (D) Peak wavelength <i>vs</i> temperature (obtained from (C)). The transition points in B) and D) are determined by sigmoidal fitting (solid curves).</p

Sequences of wild type ILPR-I4 and ILPR-I3, a scrambled sequence, and the mutants used in this study.

<p>Sequences of wild type ILPR-I4 and ILPR-I3, a scrambled sequence, and the mutants used in this study.</p

Formation of an intermolecular i-motif.

<p>(A) Schematic of the formation of an intermolecular i-motif. The proposed structure in the ILPR-I3 is shown on the left. Each C:CH⁺ pair is represented by two opposite rectangles. (B) PAGE gel image of the Br₂ footprinting experiment. Lane 1, the ILPR-I3/ILPR-I1 (I₃+I₁) mixture at pH 7.0. Lane 2, the I₃+I₁ sample at pH 5.5. Lane 3, the ILPR-I3 (I₃) at pH 5.5. Lane 4, the ILPR-I4 (I₄) at pH 5.5. The band intensity for lane 2 is shown to the left of the gel. The fold protection for the I₃+I₁ sample at pH 5.5 is shown to the right. The dotted vertical lines indicate the average fold protection for each C4 tract. The blue arrows indicate the loop cytosines. Error bar represents the standard deviation of three independent experiments. The blue arrows indicate the cytosines in the ACA section of each fragment. Note that the fold protection for adenines at 3'-end (indicated by asterisk *) is not accurate since they are close to the uncut oligo. (C) Normalized rupture force histogram for the I₃+I₁ sample at pH 5.5. The solid black curve represents a two-peak Gaussian function. The dotted curve is the Gaussian fit for the rupture force histogram of the ILPR-I3 at pH 5.5.</p

Mutation analysis in a 10 mM sodium phosphate buffer (pH 5.5) with 100 mM KCl.

<p>(A) 295 nm UV melting curves of the ILPR-I3 (“Wild Type”) and the mutants at 10 µM concentration. (B) Top panel, <i>T</i>_1/2-melt of the mutants and the ILPR-I3. “W” depicts the wild type ILPR-I3. Bottom panel, CD peak shift of the mutants and the scrambled sequence (ILPR-S3) with respect to the 285 nm peak in the ILPR-I3. The horizontal dotted lines (green) represent the average value for each C4 tract. Statistical treatment is represented by the <i>P</i> values in the bottom panel. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039271#pone-0039271-t001" target="_blank">Table 1</a> for DNA sequences.</p

Single-Molecule Measurements of the Binding between Small Molecules and DNA Aptamers

Aptamers that bind small molecules can serve as basic biosensing platforms. Evaluation of the binding constant between an aptamer and a small molecule helps to determine the effectiveness of the aptamer-based sensors. Binding constants are often measured by a series of experiments with varying ligand or aptamer concentrations. Such experiments are time-consuming, material nonprudent, and prone to low reproducibility. Here, we use laser tweezers to determine the dissociation constant for aptamer–ligand interactions at the single-molecule level from only one ligand concentration. Using an adenosine 5′-triphosphate disodium salt (ATP) binding aptamer as an example, we have observed that the mechanical stabilities of aptamers bound with ATP are higher than those without a ligand. Comparison of the change in free energy of unfolding (Δ<i><i>G</i></i>_unfold) between these two aptamers yields a Δ<i><i>G</i></i> of 33 ± 4 kJ/mol for the binding. By applying a Hess-like cycle at room temperature, we obtained a dissociation constant (<i>K</i>_d) of 2.0 ± 0.2 μM, a value consistent with the <i>K</i>_d obtained from our equilibrated capillary electrophoresis (CE) (2.4 ± 0.4 μM) and close to that determined by affinity chromatography in the literature (6 ± 3 μM). We anticipate that our laser tweezers and CE methodologies may be used to more conveniently evaluate the binding between receptors and ligands and also serve as analytical tools for force-based biosensing

Electron Microscopic Visualization of Protein Assemblies on Flattened DNA Origami

DNA provides an ideal substrate for the engineering of versatile nanostructures due to its reliable Watson–Crick base pairing and well-characterized conformation. One of the most promising applications of DNA nanostructures arises from the site-directed spatial arrangement with nanometer precision of guest components such as proteins, metal nanoparticles, and small molecules. Two-dimensional DNA origami architectures, in particular, offer a simple design, high yield of assembly, and large surface area for use as a nanoplatform. However, such single-layer DNA origami were recently found to be structurally polymorphous due to their high flexibility, leading to the development of conformationally restrained multilayered origami that lack some of the advantages of the single-layer designs. Here we monitored single-layer DNA origami by transmission electron microscopy (EM) and discovered that their conformational heterogeneity is dramatically reduced in the presence of a low concentration of dimethyl sulfoxide, allowing for an efficient flattening onto the carbon support of an EM grid. We further demonstrated that streptavidin and a biotinylated target protein (cocaine esterase, CocE) can be captured at predesignated sites on these flattened origami while maintaining their functional integrity. Our demonstration that protein assemblies can be constructed with high spatial precision (within ∼2 nm of their predicted position on the platforms) by using strategically flattened single-layer origami paves the way for exploiting well-defined guest molecule assemblies for biochemistry and nanotechnology applications