We demonstrate the use of a microfluidic stagnation point flow to trap and extend single molecules of double-stranded (ds) genomic DNA for detection of target sequences along the DNA backbone. Mutant EcoRI-based fluorescent markers are bound sequence-specifically to fluorescently labeled ds λ-DNA. The marker-DNA complexes are introduced into a microfluidic cross slot consisting of flow channels that intersect at ninety degrees. Buffered solution containing the marker-DNA complexes flows in one channel of the cross slot, pure buffer flows in the opposing channel at the same flow rate, and fluid exits the two channels at ninety degrees from the inlet channels. This creates a stagnation point at the center of a planar extensional flow, where marker-DNA complexes may be trapped and elongated along the outflow axis. The degree of elongation can be controlled using the flow strength (i.e., a non-dimensional flow rate) in the device. Both the DNA backbone and the markers bound along the stretched DNA are observed directly using fluorescence microscopy and the location of the markers along the DNA backbone is measured. We find that our method permits detection of each of the five expected target site positions to within 1.5 kb with standard deviations of <1.5 kb. We compare the method’s precision and accuracy at molecular extensions of 68% and 88% of the contour length to binding distributions from similar data obtained via molecular combing. We also provide evidence that increased mixing of the sample during binding of the marker to the DNA improves binding to internal target sequences of dsDNA, presumably by extending the DNA and making the internal binding sites more accessible.

We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzyme's binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker's ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods.

Site-directed spin labeling, wherein a nitroxide side chain is introduced into a protein at a selected mutant site, is increasingly employed to investigate biological systems by electron spin resonance (ESR) spectroscopy. An understanding of the packing and dynamics of the spin label is needed to extract the biologically relevant information about the macromolecule from ESR measurements. In this work, molecular dynamics (MD) simulations were performed on the spin labeled restriction endonuclease, EcoRI in complex with DNA. Mutants of this homodimeric enzyme were previously constructed and distance measurements were performed using the Double Electron Electron Resonance experiment. These correlated distance constraints have been leveraged with MD simulations to learn about side chain packing and preferred conformers of the spin label on sites in an α-helix and a β-strand. We found three dihedral angles of the spin label side chain to be most sensitive to the secondary structure where the spin label was located. Conformers sampled by the spin label differed between secondary structures as well. Cα-Cα distance distributions were constructed and used to extract details about the protein backbone mobility at the two spin labeled sites. These simulation studies enhance our understanding of the behavior of spin labels in proteins and thus expand the ability of ESR spectroscopy to contribute to knowledge of protein structure and dynamics.

KeywordsEPR spectroscopy; DEER; DNA binding protein; structure elucidation; protein flexibility; noncognate complexIn this communication, we show that the EcoRI restriction endonuclease binds different classes of DNA sites in the same binding cleft. EcoRI generates widespread interest because it exhibits an extraordinary sequence selectivity to carry out its function of cleaving incoming foreign DNA without causing potentially lethal cleavage of cellular DNA. For example, EcoRI binds to its correct recognition site GAATTC up to 90,000-fold better than to miscognate sites that have one incorrect base pair. [1,2] The ∼650 specific sites in the E. coli genome are protected from cleavage by double-strand methylation. The ∼21,000 miscognate sites are not methylated, but are still cleaved by EcoRI with a second-order rate constant that is ∼10 9 -fold lower.[1,2] EcoRI forms only non-specific complexes with no cleavage at sites that differ from GAATTC by two or more base pairs. [1,2] In order to understand the source of such high specificity, it is necessary to determine how the structures of EcoRI complexes differ at specific, miscognate (5/6 bp match), and nonspecific (≤4/6 bp match) DNA sites. This effort is timely given the extensive genetic, biochemical and biophysical data on EcoRI.[1-9] Footprinting results [1] suggest that the three classes of complexes are "structurally" distinct, and thermodynamic profiles (ΔG°, ΔH°, ΔS°, ΔC°P) [3,4] suggest that the specific complex has more restricted conformationalvibrational mobility of the protein and DNA. There are crystal structures of the free protein, Figure 1 shows the structure of the EcoRI specific complex. [6,7] The protein contains a large, relatively rigid and structured globular "main" domain and a smaller "arm" region. The protein arms are invisible in the free protein[6] but become ordered and enfold the DNA in the specific complex, where they play a role in modulating specificity.[2,4] Mutations R131C, S180C, and K249C-S180C were chosen based on the crystal structure. [6,7] These sites are solvent accessible and therefore likely to spin label with minimal perturbation to protein structure. Residues R131 and S180 lie in the inner and outer arms, respectively. Residue K249 is in the main domain, which has very restricted movement [6] and acts as a reference point. Since EcoRI is a 62 kDa homodimer, single cysteine mutations provide two sites for spin labeling, and double mutations provide four sites.The proteins were spin labeled at the cysteines with the methanethiosulfonate spin label (MTSSL). There is an intrinsic cysteine at position 218, but it is buried, leading to <10% labeling even with a 100-fold molar excess of the spin label. The mutant proteins and their spin labeled derivatives catalyze DNA cleavage and have DNA binding affinities similar to that of wild type EcoRI, indicating that they are functionally active (Supporting Information).DEER experiments [11] were performed on spin labeled S180C specific and non-specific complexes, and on R131C and...