Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Magnesium-induced folding of SL ribozyme constructs. Peak E shifts from the Gaussian fits of the FRET histograms at Mg2+ concentrations from 0.01 mM to 1 M for the SL (▪) and WT (⋄) 2WJ, 3WJ, and 4WJ ribozymes (parts A, B, and C, respectively). Peak E values for a control donor-acceptor labeled DNA molecule with dyes separated by 14 base pairs are shown for comparison as solid triangles in the 3WJ graph in part B.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Control, Labeling, Comparison

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Ratiometric spFRET data for the WT 4WJ ribozyme. (A) Example of donor (black) and acceptor (gray) emission bursts observed in spFRET experiments. A time trace is presented for the WT 4WJ ribozyme at a concentration of 100 pM in 50 mM Tris/HCl buffer with 0.1 mM Mg2+ at room temperature, and the data integration time per point was 0.5 ms. Fluorescent donor and acceptor bursts are observed above background. A buffer (background) time trace is also shown for comparison. Only bursts above a threshold of 40 counts (sum of donor and acceptor signals) were used to construct FRET efficiency histograms. (B) FRET efficiency histogram derived from calculation of FRET efficiencies from each accepted donor/acceptor event above a threshold of 40 counts, showing the docked and undocked subpopulations (see text), as well as the zero-E peak. The solid lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Concentration Assay, Comparison, Construct, Derivative Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: FRET efficiency histogram for a G11I mutant of the 4WJ ribozyme at 0.1 mM Mg2+, using the same conditions as for data presented in Fig. 3. Solid lines show fits using Gaussian functions. The relative area of the high efficiency (docked) peak is markedly reduced relative to the wild-type 4WJ ribozyme (Fig. 3), reflecting a destabilized tertiary structure for the mutant.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Mutagenesis

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Schematic representation of WT ribozyme constructs used for spFRET studies (upper panel). FRET efficiency histograms (bars) for wild-type 2WJ, 3WJ, and 4WJ hairpin ribozymes, showing undocked and docked ribozyme subpopulations at Mg2+ concentrations of 0.01, 0.1, 1, and 10 mM (lower panel). Data were acquired using a ribozyme concentration of 100 pM in 50 mM Tris/HCl buffer with Mg2+ at room temperature, and the data integration time per point was 0.5 ms. The smooth lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Concentration Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Single-loop 2WJ, 3WJ, and 4WJ ribozyme constructs (upper panel) and corresponding FRET efficiency histograms at Mg2+ concentrations of 0.01, 0.1, 1, and 10 mM (lower panel). Data were acquired using a ribozyme concentration of 100 pM in 50 mM Tris/HCl buffer with Mg2+ at room temperature, and the data integration time per point was 0.5 ms. The smooth lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Concentration Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Kinetic information from peak shape analysis of spFRET histograms. (A) Overlay of FRET efficiency histograms for SL (bold solid line) and WT 4WJ (data as bar graph, Gaussian fit as solid line) ribozymes at 10 mM Mg2+, emphasizing the asymmetric peak shape for the SL 4WJ. The error bars represent single standard deviations. (B) Overlay of FRET histograms from two-state simulations (lines) and experimental data (bars; errors represent single standard deviations) for the SL 4WJ at 10 mM Mg2+. The extended-undocked (EU)–quasi-docked (QD) equilibrium scheme is shown in the inset. Simulated histograms are shown for forward/reverse (k1/k−1) rate constants of 20 × 103/6.8 × 103 s−1 (bold solid line, 1), 41 × 103/14 × 103 s−1 (bold dashed line, 2), and 10 × 103/3.4 × 103 s−1 (bold dash-dot-dashed line, 3) with a corresponding equilibrium constant of 3 in favor of the quasi-docked state in all cases. See text for details.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques:

Journal: Journal of Chemical Information and Modeling

Article Title: SILVR: Guided Diffusion for Molecule Generation

doi: 10.1021/acs.jcim.3c00667

Figure Lengend Snippet: Schematic of the equivariant diffusion model with selective iterative latent variable refinement (SILVR) indicated for every denoising step. Here, the reference in blue on the left shows 3 small fragments. They evolve over time t in the diffusion process to resemble a Gaussian distribution at t = T , see eq . The β represents the noise added at each step, and the dots show the steps omitted from time t = 3 to t = T . As atoms effectively “diffuse”, they can be perceived as changing position. To generate a new sample, a sample is generated from p θ ( x ) according to eq , this distribution is from the learned EDM. At each denoising step, a set of reference fragments ( y t ) at that same level of noise t is used, which is indicated by the SILVR arrows to condition the EDM. This is controlled through SILVR at a given rate r S , until a new sample that resembles the reference is generated (following the bottom row along the yellow boxes and EDM arrows).

Article Snippet: The agreement in the shape of the samples and the binding site of MPro were determined using the OpenEye toolkit version 2022.2.2 Gaussian scoring function Shapegauss., This scoring function measures the shape complementarity between the ligand and receptor by considering each heavy atom as a Gaussian function.

Techniques: Diffusion-based Assay, Generated

Journal: Journal of Chemical Information and Modeling

Article Title: SILVR: Guided Diffusion for Molecule Generation

doi: 10.1021/acs.jcim.3c00667

Figure Lengend Snippet: Validation measures of the SILVR model using fragments x0072 and x0354 as reference coordinates. (A) Ratio of stable atoms—an atom is determined as stable if the valence matches the expected valence for the element. (B) RMSD from reference—the calculated RMSD between the reference and sample, using an absolute one-to-one mapping ignores atom identity with low RMSD meaning molecules are similar to the reference and high RMSD they are not. (C) OpenEye measure Shapegauss—a Gaussian scoring function describing the shape fit between Mpro and samples, ignoring chemical interactions. A lower score means a better shape fit of the molecule. (D) Geometry stability—AIMNet geometry optimization was completed with Auto3D using the SMILES string of each sample. RMSD was calculated between the predicted geometry and the sampled geometry using RDKit. Horizontal lines indicate the sample median and circles indicate the sample mean.

Article Snippet: The agreement in the shape of the samples and the binding site of MPro were determined using the OpenEye toolkit version 2022.2.2 Gaussian scoring function Shapegauss., This scoring function measures the shape complementarity between the ligand and receptor by considering each heavy atom as a Gaussian function.

Techniques: Biomarker Discovery