prism-type tirf microscope nikon eclipse ti2  (Nikon)

Journal: Nucleic Acids Research

Article Title: Single-molecule imaging reveals a direct role of CTCF’s zinc fingers in SA interaction and cluster-dependent RNA recruitment

doi: 10.1093/nar/gkae391

Figure Lengend Snippet: CTCF enriches on both, single and 4× CTCF-binding sites, with a lifetime of ∼50 min. ( A ) Schematic representation of CTCF containing the 11 ZFs, the RNA recognition motif (RBR), the elongated termini and the CTCF binding site (CBS) containing core and upstream motif. ( B ) Schematic representation of the DNA curtains assay. The DNA substrate was designed with either two cassettes of 4x CBSs (light green) with opposing orientation or one cassette with 1× CBS that were included into λ-DNA (black), which was tethered between Cr barriers (grey) on a custom-built flow cell. ( C ) Representative images of TIRF microscopy of 10 nM AF-568-CTCF-WT binding on DNA including either 1× (left) or 4× (right) CTCF binding sites (CBSs, indicated by green arrows). CTCF was first loaded at 50 mM NaCl leading to full coverage of the DNA (top). After washing with 300 mM NaCl, CTCF is enriched on the binding sites (middle) and remains bound for a long time (bottom). ( D ) Representative kymogram of CTCF binding to 1× (top) or 4× (middle) CBSs. Kymogram is shown after a 300 mM NaCl wash. Some non-CBS-bound CTCF diffuse (bottom). ( E ) CTCF enriches at 1× ( N = 477) and 4× CBSs ( N = 427) after a high salt wash. Light green bars indicate position of CBSs. A/T-content of the DNA substrate is shown with the black line. ( F ) CTCF is more strongly enriched (16×) on a 4× than on a 1× site (7×). ( G ) Lifetimes on 1× and 4× CBSs measured at different laser frame delay and 100 ms illumination time. A global fit was applied to correct for photobleaching and the presence of multiple CTCFs on the 4× CBSs. ( H ) Photobleaching-corrected CTCF lifetimes on 1× CBS and 4× CBSs are similar, but significantly higher than on λ-DNA.

Article Snippet: DNA curtain experiments were carried out as described previously ( ) on a prism-type TIRF microscope (Nikon Eclipse Ti2), equipped with three illumination lasers (488, 561 and 640 nm Coherent OBIS), an electron multiplying charged coupled camera (iXon Life, Andor) and a syringe-pump-driven microfluidics system supplying the sample chamber.

Techniques: Binding Assay, Microscopy

Journal: Nucleic Acids Research

Article Title: Single-molecule imaging reveals a direct role of CTCF’s zinc fingers in SA interaction and cluster-dependent RNA recruitment

doi: 10.1093/nar/gkae391

Figure Lengend Snippet: CTCF ZFs stabilize SA on CBSs ( A ) TIRF microscopy at 100 ms illumination time of fluorescently labeled CTCF (green) and SA (pink) preincubated (10 nM AF568-CTCF with 100 nM SA-LD655) before loading on DNA curtains, before (top) and after (bottom) 300 mM NaCl wash. ( B ) Histograms of CTCF (green, left: N = 762; right: N = 747), SA1 (purple, N = 299) and SA2 (pink, N = 196) binding positions after simultaneous load and salt wash. CBSs are shown as green bars and the AT-ratio as a black line. ( C ) TIRF microscopy of 10 nM AF568-CTCF after salt wash (top) followed by 100 nM SA-LD655 load (bottom). Color code as in (A). ( D ) Histograms of CTCF (green, left: N = 171; right: N = 120), SA1 ( N = 380) and SA2 ( N = 80) binding positions after sequential load. ( E ) TIRF microscopy of 100 nM SA-LD655 (magenta) binding on DNA-curtains. ( F ) Histograms of SA1 ( N = 1321) and SA2 ( N = 875) binding positions on λ-DNA. ( G ) Lifetime of SAs on AT-rich and GC-rich DNA regions at 50 and 150 mM NaCl. At 50 mM NaCl, both SAs have a higher lifetime on AT-rich than on GC-rich regions. At 150 mM, only SA1 shows a higher lifetime on AT-rich, which is also significantly higher than for SA2. ( H ) SA1 and SA2 diffusion coefficients on AT-rich and GC-rich regions (coloring like in G). SA1 and SA2 diffuse significantly faster on GC-rich than on AT-rich regions. SA2 diffuses significantly faster than SA1 on GC-rich regions, but not on AT-rich regions. ( I ) Lifetime of SAs in absence of CTCF and after recruitment to CBSs by different CTCF constructs. SA1 and SA2 have a significantly higher lifetime on CTCF WT than on DNA. Both SAs have similar lifetimes on CTCF variants compared to CTCF WT.

Article Snippet: DNA curtain experiments were carried out as described previously ( ) on a prism-type TIRF microscope (Nikon Eclipse Ti2), equipped with three illumination lasers (488, 561 and 640 nm Coherent OBIS), an electron multiplying charged coupled camera (iXon Life, Andor) and a syringe-pump-driven microfluidics system supplying the sample chamber.

Techniques: Microscopy, Labeling, Binding Assay, Diffusion-based Assay, Construct

Journal: Nucleic Acids Research

Article Title: Single-molecule imaging reveals a direct role of CTCF’s zinc fingers in SA interaction and cluster-dependent RNA recruitment

doi: 10.1093/nar/gkae391

Figure Lengend Snippet: RNA recruitment by oligomeric CTCF. ( A ) 100 bp Cy3-UTP labeled RNA-loading (25 ng/μl) on CBS-enriched Alexa-Fluor 660-CTCF. Left: Experimental workflow. Right: TIRF microscopy at 100 ms illumination time of CBS-bound CTCF (green) before RNA load (top) and of Cy3-UTP labeled RNA (cyan) after RNA load (bottom). No RNA capture was observed. ( B ) Representative intensity curve of a two-step CTCF photobleaching event. Bleaching steps are illustrated by a black line. ( C ) Histogram of CTCF bleaching steps on 4× CBSs. Same as , added here for comparison. ( D ) 100 bp Cy3-UTP labeled RNA-loading (25 ng/μl) on clustered Alexa-Fluor 660-CTCF. Left: Experimental workflow. Right: TIRF microscopy at 100 ms illumination time of CTCF clusters (green) before RNA load (top) and of Cy3-UTP labeled RNA (cyan) after RNA load. RNA is recruited to CTCF clusters (white arrows). ( E ) Representative intensity curve of multi-step CTCF bleaching event of a CTCF-RNA cluster. Steps are illustrated by a black line. ( F ) Histogram of CTCF bleaching steps in CTCF-RNA clusters. ( G ) 10 nM ATTO-643-CTCF loading at RNA transcripts. Left: Experimental workflow. RNA transcripts were formed by loading T7-Pol and facilitating transcription as before. Right: TIRF microscopy at 100 ms illumination time of Cy3-UTP labeled RNA in RNA transcripts (cyan) before CTCF load (top) and of CTCF (green) after loading (bottom). CTCF is partially recognizing RNA transcripts (orange arrows), but is mainly coating the DNA (white arrows). ( H ) Enrichment of CTCF on 4× CBSs is not significantly different in presence or absence of RNA transcripts. ( I ) CTCF lifetime on RNA transcripts is similar to lifetimes on λ-DNA and significantly smaller than on 4× CBSs.

Article Snippet: DNA curtain experiments were carried out as described previously ( ) on a prism-type TIRF microscope (Nikon Eclipse Ti2), equipped with three illumination lasers (488, 561 and 640 nm Coherent OBIS), an electron multiplying charged coupled camera (iXon Life, Andor) and a syringe-pump-driven microfluidics system supplying the sample chamber.

Techniques: Labeling, Microscopy, Comparison