A Spectroscopic Approach to Unravel the Local Conformations of a G‑Quadruplex Using CD-Active Fluorescent Base Analogues

The formation of a stable G-quadruplex (GQ) can inhibit the increased telomerase activity that is common in most cancers. The global structure and the thermal stability of the GQs are usually evaluated by spectroscopic methods and thermal denaturation properties. However, most biochemical processes involving GQs might require local conformational changes at the guanine tetrad (G4) level. These local conformational changes of individual G4 layers during protein and drug interactions have not yet been explored in detail. In this study, we monitored the local conformations of individual G4 layers in GQs using 6-methylisoxanthopterine (6MI) chromophores, which are circular dichroism (CD)-active fluorescent base analogues of guanine, as local conformational probes. A synthetic, tetramolecular, parallel GQ with site-specifically positioned 6MI monomers or dimers was used as the experimental construct. Analytical ultracentrifugation studies and gel electrophoretic studies showed that properly positioned 6MI monomers and dimers could form stable GQs with CD-active fluorescent G4 layers. The local conformation of individual fluorescent G4 layers in the GQ structure was then tracked by monitoring the absorbance, fluorescence intensity, thermal melting, fluorescence quenching, and CD changes of the incorporated probes. Overall, these studies showed that site-specifically incorporated fluorescent base analogues could be used as probes to monitor the local conformational changes of individual G4 layers of a GQ structure. This method can be applied to explore the details of small molecule–GQ interaction at the level of the individual G4 layers, which may prove to be useful in designing drugs to treat GQ-related genetic disorders, cancer, and aging

A Single-Molecule View of the Assembly Pathway, Subunit Stoichiometry, and Unwinding Activity of the Bacteriophage T4 Primosome (helicase–primase) Complex

Single-molecule fluorescence resonance energy transfer (smFRET) methods were used to study the assembly pathway and DNA unwinding activity of the bacteriophage T4 helicase–primase (primosome) complex. The helicase substrates used were surface-immobilized model DNA replication forks “internally” labeled in the duplex region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in the lagging and leading strands. The time dependence of the smFRET signals was monitored during the unwinding process, and helicase rates and processivities were measured as a function of GTP concentration. This smFRET approach was also used to investigate the subunit stoichiometry of the primosome and the assembly pathway required to form functional and fully active primosome–DNA complexes. We confirmed that gp41 helicase monomer subunits form stable hexameric helicases in the presence of GTP and that the resulting (gp41)₆ complexes bind only weakly at DNA fork junctions. The addition of a single subunit of gp61 primase stabilized the resulting primosome complex at the fork and resulted in fully active and processive primosome helicases with gp41:gp61 subunit ratios of 6:1, while higher and lower subunit ratios substantially reduced the primosome unwinding activity. The use of alternative assembly pathways resulted in a loss of helicase activity and the formation of metastable DNA–protein aggregates, which were easily detected in our smFRET experiments as intense light-scattering foci. These single-molecule experiments provide a detailed real-time visualization of the assembly pathway and duplex DNA unwinding activity of the T4 primosome and are consistent with more indirect equilibrium and steady state results obtained in bulk solution studies

Examples for the determination of radial magnification errors.

<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p

Absence of a long-term trend in <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values of the BSA monomer with time of experiment for the three kits (blue, green, and magenta).

<p>Highlighted as bold solid line is the overall average, and the grey area indicates one standard deviation.</p

Corrected best-fit apparent monomer molecular mass from integration of the <i>c</i>(<i>s</i>) peak when scanned with the absorbance system (green) and the interference system (magenta).

<p>Only data with rmsd less than 0.01 OD or 0.01 fringes were included. The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.</p

Analysis of the rotor temperature.

<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p

Examples of transient changes in the console temperature reading during the SV experiment, as saved in the scan file data.

<p>For comparison, the maximum adiabatic cooling of -0.3°C would be expected after approximately 300 sec, recovering to the equilibrium temperature after approximately 1,200 s (see Fig 3 in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref033" target="_blank">33</a>]).</p

Histogram and box-and-whisker plot of <i>s</i>-values of the BSA monomer after different corrections: Raw experimental <i>s</i>-values (black, with grey histogram), scan time corrected <i>s</i>_<i>t</i>-values (blue), rotor temperature corrected <i>s</i>_<i>20T</i>-values (green), or radial magnification corrected <i>s</i>_<i>r</i>-values (cyan), and fully corrected <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values (red with red histogram).

<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as a vertical line.</p

Example for the analysis of absorbance data from the sedimentation velocity experiment of BSA.

<p>(A) Absorbance scans (symbols) and best-fit <i>c</i>(<i>s</i>) model at different points in time indicated by color temperature. (B and C) Bitmap and overlay of the residuals of the fit. (D) <i>c</i>(<i>s</i>) sedimentation coefficient distribution showing peaks for monomer, dimer, trimer, and traces of higher oligomers.</p

Distributions of calculated BSA monomer signals for the different kits and the different optical systems.

<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as vertical line.</p