Journal: bioRxiv

Article Title: Modulating cell-free transcription electrolytically with switchable DNA triplexes

doi: 10.1101/2025.04.25.650642

Figure Lengend Snippet: (a) aPCR workflow. The sequences for the EDGE construct with and without tail are synthesised as dsDNA plasmids, then amplified to form a linear dsDNA template via PCR with one of the primers being biotinylated, depending on which strand (‘top’ – blue, right hand side; ‘bottom’ – orange, left hand side) is being produced. The biotinylated dsDNA template is then used for aPCR, with a single primer, to generate ssDNA encoding one of the strands used to produce the final EDGE construct. The biotinylated dsDNA template is then removed via a streptavidin pulldown binding to the biotin and removing it from solution, leaving a purified ssDNA product. The ssDNA products for top and bottom strand are then hybridised. (b) Agarose gel electrophoresis (3%, 120V, 50 minutes run time with ice cooling, GelRed staining) of ssDNA extracted from a preceding gel confirming that all strands are the correct size and hybridise as expected. (c) Fluorescence intensity after 1000 minutes of transcription at pH8 for the original TFR-only (non-triplex forming control), T7D and T7P constructs. (d) Transcription kinetics for the three constructs at six different pHs. For (c) and (d): each data point is the mean of three replicates and the error bar is the standard deviation. Buffer = 1x 100% AceOTB.

Article Snippet: Additionally, ssDNA used during the small-scale chemical control tests were not purified via streptavidin pulldown, but were separated via agarose gel electrophoresis, excised and purified using a gel extraction kit (NEB - #T1020S).

Techniques: Construct, Amplification, Produced, Binding Assay, Purification, Agarose Gel Electrophoresis, Staining, Fluorescence, Control, Standard Deviation

Journal: Cell Reports

Article Title: TSPAN5 Enriched Microdomains Provide a Platform for Dendritic Spine Maturation through Neuroligin-1 Clustering

doi: 10.1016/j.celrep.2019.09.051

Figure Lengend Snippet: TSPAN5 Promotes NLG1 Clustering (A) Confocal images of dendrites from DIV 12 rat hippocampal cultured neurons transfected at DIV 5 with AP-NLG1, BirA-ER, and scrambled, Sh-TSPAN5, rescue, DLQ, or NIYF all co-expressing mCherry. Surface-applied Streptavidin-488 clusters shown in green. Scale bar, 5 μm. (B) Quantification of the cluster density (clusters/square micrometer) and average size (square micrometers) of Streptavidin-488 clusters. Scrambled, n = 19 neurons; Sh-TSPAN5, n = 20 neurons; rescue, n = 18 neurons; DLQ, n = 27 neurons; NIYF, n = 27 neurons. (C) Single-molecule tracking experiment with mStrav-647. Images of DIV 12 rat hippocampal cultured neurons transfected at DIV 5 with AP-NLG1, BirA-ER, and scrambled, Sh-TSPAN5, rescue, or PLM all co-expressing GFP. Top panels: widefield GFP signal of dendrites. Middle panels (intensity): super-resolved mStrav-647 detection maps as an intensity scale. Bottom panels (tracks): mStrav-647 trajectories shown in pseudocolors. Scale bar, 2 μm. (D) Quantification of single-molecule tracking. Left panel: quantification of global diffusion coefficient (square micrometers per second). Right panel: logarithmic distribution plot of diffusion coefficients of scrambled- (black), Sh-TSPAN5- (blue), rescue- (purple), or PLM-transfected neurons (green). Scrambled, n = 16 neurons; Sh-TSPAN5, n = 17 neurons; rescue, n = 16 neurons; PLM, n = 12 neurons. (E) Confocal images of DIV 14 cultured rat hippocampal neurons transfected at DIV 5 with HA-tagged NLG1 and either GFP-expressing scrambled or Sh-TSPAN5 constructs. Neurons treated from DIV 12 to 14 with either non-clustered neurexin1β-Fc (left panels) or neurexin1β-Fc pre-clustered with an unlabeled α-Fc antibody (right panels). Surface-applied α-HA antibody shown in red; surface-applied Alexa 647-labeled α-Fc shown in blue. Purple arrowheads show mushroom spines positive for clusters. (F) Pie charts of the proportion of stubby, thin, or mushroom spines relative to (E). Exact values are shown in . See also . Graphs represent mean ± SEM. In (F), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus scrambled non-clustered; °p < 0.05, °p < 0.01, °p < 0.001 versus Sh-TSPAN5 non-clustered.

Article Snippet: Transfected cells used for streptavidin pulldown experiments were grown for 48 h in Neurobasal (GIBCO) medium supplemented with 2% B27 (prepared as in ), 0.25% L-glutamine and 1% penicillin/streptamycin (Invitrogen).

Techniques: Cell Culture, Transfection, Expressing, Diffusion-based Assay, Construct, Labeling

Journal: Cell Reports

Article Title: TSPAN5 Enriched Microdomains Provide a Platform for Dendritic Spine Maturation through Neuroligin-1 Clustering

doi: 10.1016/j.celrep.2019.09.051

Figure Lengend Snippet:

Article Snippet: Transfected cells used for streptavidin pulldown experiments were grown for 48 h in Neurobasal (GIBCO) medium supplemented with 2% B27 (prepared as in ), 0.25% L-glutamine and 1% penicillin/streptamycin (Invitrogen).

Techniques: Transduction, Recombinant, Modification, Software, Imaging

Journal: Nature chemistry

Article Title: Sequencing abasic sites in DNA at single-nucleotide resolution

doi: 10.1038/s41557-019-0279-9

Figure Lengend Snippet: Chemical tagging of DNA abasic sites. a ) Structures of naturally occurring aldehyde residues found in DNA. b ) Structure of HIPS probe 1 . c ) Stability of AP-ODN and probe 1 -labelled ODNs after biotinylation, to alkaline-cleavage (100 mM NaOH, 15 min). Reactions were carried out at 70°C except where labelled RT, where room temperature was used. 5-fC-1-biotin was obtained by extending the reaction time with 1 to 24 h. Reactions were followed by LC-MS. Results from three independent replicates are shown. d ) Workflow of chemical enrichment of abasic sites using probe 1 . Tagging of aldehyde residues followed by biotinylation through CuAAC and streptavidin pulldown enriches for all reacted aldehydes, whilst the removal of the biotin tag by alkaline-cleavage occurs only at AP sites. The truncated DNA fragments with 5’-phosphate termini are then recovered and prepared for next-generation sequencing. Sequencing reads stack up immediately after captured AP sites due to the site-specific DNA cleavage step via base-catalyzed elimination.

Article Snippet: Streptavidin pulldown: Magnesphere streptavidin beads (Promega, 50 μL) were pre-washed three times with 1X binding buffer (5 mM Tris pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20) and resuspended in 50 μL of 2X binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.1% Tween 20).

Techniques: Liquid Chromatography with Mass Spectroscopy, Next-Generation Sequencing, Sequencing

Journal: Nature chemistry

Article Title: Sequencing abasic sites in DNA at single-nucleotide resolution

doi: 10.1038/s41557-019-0279-9

Figure Lengend Snippet: Outline of snAP-seq and analysis of synthetic DNA spike-ins. a ) Enrichment of AP DNA relative to GCAT, 5-fC and 5-fU DNA, after first streptavidin enrichment. DNA was recovered under alkaline-cleavage conditions (100 mM NaOH, 70°C), before neutralization and purification. DNA was quantified by qPCR, and normalized to input DNA. The recovery of each DNA sequence was compared to AP DNA, and represented as a fold-enrichment of AP DNA. Apart from GCAT DNA, all primers were designed 3’- to each modification and therefore expected cleavage site to allow DNA amplification. Mean ± S.E.M. of three replicates are shown. b ) Recovery of DNA sequences after second streptavidin enrichment. AP DNA remains unbound and is recovered from the supernatant, whilst 5-fU DNA is captured on the beads. DNA was quantified by qPCR and normalized to the amount of DNA recovered after first streptavidin enrichment. Results from three replicates are plotted. c ) Library-preparation workflow of snAP-seq. d ) Distribution of reads aligning to each model DNA sequence after next-generation sequencing. The number of reads of each ODN was normalized to the corresponding reverse strand in the input sample (see ). Only the modification-containing, forward strands of each ODN are analyzed.

Article Snippet: Streptavidin pulldown: Magnesphere streptavidin beads (Promega, 50 μL) were pre-washed three times with 1X binding buffer (5 mM Tris pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20) and resuspended in 50 μL of 2X binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.1% Tween 20).

Techniques: Neutralization, Purification, Sequencing, Modification, DNA Amplification, Next-Generation Sequencing