Brown Nat Protocols Fig 2

Figure 2 | Comparison of hybridization strategies. a, Hybridization efficiencies plotted as the percentage of signal scored (mean with SD) in C127 cells for probes pEx and pCx36 (red and green respectively). As a lack of hybridization signals was occasionally noted in RASER-FISH samples, owing to incomplete BrdU labelling, we assessed hybridization efficiency as follows: on an allelic basis, the presence of a single signal (either green or red) deemed that allele as scorable. At that same allele, the presence (or absence) of the neighbouring signal was recorded. Hybridization frequencies were expressed as number of alleles with both red and green signals / number of all scorable alleles ×100. The hybridization frequencies shown are calculated from 501 (4 min), 715 (4 min+dry), 755 (RASER) and 218 (30 min+dry) alleles. b, Comparison of single-strandedness and nuclear integrity. Single-stranded DNA detected by antibody in example HeLa and C127 cells after simple immunofluorescence (IF control) or the three FISH methods, 4 min, 4 min+dry, RASER. Upper panels show the ssDNA antibody labelling signal (green/white). Scale bar, 5 µm. Lower panels show an expanded view of the nuclear periphery of the same cells with the ssDNA signal (green) against a DAPI counterstain (purple). The disruption to nuclear integrity in the heat-denatured samples is evident. Scale bar, 1 µm. c, Comparison of access to blocks of DNA repeats. Example C127 nuclei hybridized by the three FISH methods with a probe to gamma satellite DNA repeats (green) against a DAPI counterstain, all imaged with the same settings. RASER-FISH provides the most comprehensive labelling. Scale bar, 5 µm.</p

Brown Nat Protocols Fig 2b

2b, Comparison of single-strandedness and nuclear integrity. Single-stranded DNA detected by antibody in example HeLa and C127 cells after simple immunofluorescence (IF control) or the three FISH methods, 4 min, 4 min+dry, RASER. Upper panels show the ssDNA antibody labelling signal (green/white). Scale bar, 5 µm. Lower panels show an expanded view of the nuclear periphery of the same cells with the ssDNA signal (green) against a DAPI counterstain (purple). The disruption to nuclear integrity in the heat-denatured samples is evident. Scale bar, 1 µm

Brown Nat Protocols Fig 2c

2c, Comparison of access to blocks of DNA repeats. Example C127 nuclei hybridized by the three FISH methods with a probe to gamma satellite DNA repeats (green) against a DAPI counterstain, all imaged with the same settings. RASER-FISH provides the most comprehensive labelling. Scale bar, 5 µm.</p

Brown Nat Protocols Fig 2a

Figure 2 | Comparison of hybridization strategies. a, Hybridization efficiencies plotted as the percentage of signal scored (mean with SD) in C127 cells for probes pEx and pCx34 (red and green respectively). As a lack of hybridization signals was occasionally noted in RASER-FISH samples, owing to incomplete BrdU labelling, we assessed hybridization efficiency as follows: on an allelic basis, the presence of a single signal (either green or red) deemed that allele as scorable. At that same allele, the presence (or absence) of the neighbouring signal was recorded. Hybridization frequencies were expressed as number of alleles with both red and green signals / number of all scorable alleles ×100. The hybridization frequencies shown are calculated from 501 (4 min), 715 (4 min+dry), 755 (RASER) and 218 (30 min+dry) alleles

Brown Nat Protocols Fig 3b

Figure 3 | Comparison of chromatin structure and TAD shapes. Super-resolution 3D-SIM imaging of human RPE-1 cells after a control IF treatment or indicated FISH protocol using a KIF23 TAD-specific probe. b, Exemplary TAD FISH signals selected from several cells highlight more defined and distinct edges of the TAD signal after RASER detection compared to 4 min, 4 min+dry (bottom row of middle section) and 30 min+dry heat denaturation. Images show false colour representations of maximum intensity projections. Scale bar, 1 µm

Brown Nat Protocols Fig 6

Figure 6 | RASER-FISH hybridization examples with inset magnifications. a, Deconvolved widefield image of plasmids pCx (green) and pEx (red) from the α-globin gene region36 hybridized to C127 nucleus detected with DAPI. b, Fosmids recognising NKX2 (green) and Pax2 (red) hybridized to mouse ES cells39 with nuclei detected with DAPI. c, BAC RP24-217l105 (red) hybridized to C127 nucleus with chromatin stained with SYTOX Green (grey), imaged by 3D-SIM. Orthogonal (top) and lateral (bottom) cross sections are shown. d, 3D-SIM image of a C127 nucleus hybridized with 6 pools of oligonucleotide probes directly labelled with Atto 565, Abberior Star Red and Oregon Green and covering 1030 kb of the α-globin gene region37. Maximum projection with the nuclear boundary defined by DAPI outlined (DAPI not shown). e, 3D-STED image (maximum intensity projection) of a mouse erythroblast nucleus hybridized with 3 pools of oligonucleotide probes directly labelled with Atto 565, Abberior Star Red and Oregon Green and covering 78.5 kb of the α-globin gene region36. (f) RNA-DNA RASER-FISH image of a mouse erythroblast showing the α-globin genes detected with plasmid pA36 (red) against nascent α-globin transcripts54 (green) imaged by widefield deconvolution (maximum intensity projection). g-i, Immuno-RASER-FISH examples imaged by widefield deconvolution. g, Antibody detection of HP1α (red) combined with a plasmid probe pCx (green) (Brown 2018) detected in a DAPI-stained C127 cell nucleus (maximum intensity projection covering the central region). h, Immuno-RASER-FISH image of an antibody to fibrillarin (detecting nucleoli) (red) combined with a plasmid probe pCx36 (green) detected in a DAPI-stained C127 cell nucleus. i, Immuno-RASER-FISH image of an antibody to 53BP1 (red) combined with a BAC RP11-347N18 probe partly covering the KIF23 TAD38 (green) detected in a DAPI-stained U2OS cell nucleus (maximum intensity projection covering the central region). Scale bars are 5 µm and 1 µm (insets).</p

Brown Nat Protocols Fig 3

Figure 3 | Comparison of chromatin structure and TAD shapes. Super-resolution 3D-SIM imaging of human RPE-1 cells after a control IF treatment or indicated FISH protocol using a KIF23 TAD-specific probe. a, Central mid-sections of SYTOX Green stained nuclei show a rather inhomogeneous distribution of chromatin with strings of domain-like features separated by distinctive interchromatin space in both the IF control and after RASER-FISH. In contrast, 4 min and 30 min+dry nuclei show both a more homogeneous chromatin distribution with much reduced interchromatin regions. Insets are pseudocoloured for intensity and demonstrate the retention of interchromatin space in the RASER-FISH sample. Scale bar, 5 µm and 1 µm (insets). b, Exemplary TAD FISH signals selected from several cells highlight more defined and distinct edges of the TAD signal after RASER detection compared to 4 min, 4 min+dry (bottom row of middle section) and 30 min+dry heat denaturation. Images show false colour representations of maximum intensity projections. Scale bar, 1 µm.</p

Brown Nat Protocols Fig 3a

Figure 3 | Comparison of chromatin structure and TAD shapes. Super-resolution 3D-SIM imaging of human RPE-1 cells after a control IF treatment or indicated FISH protocol using a KIF23 TAD-specific probe. a, Central mid-sections of SYTOX Green stained nuclei show a rather inhomogeneous distribution of chromatin with strings of domain-like features separated by distinctive interchromatin space in both the IF control and after RASER-FISH. In contrast, 4 min and 30 min+dry nuclei show both a more homogeneous chromatin distribution with much reduced interchromatin regions. Insets are pseudocoloured for intensity and demonstrate the retention of interchromatin space in the RASER-FISH sample. Scale bar, 5 µm and 1 µm (insets). </p

Pd(0)/Cu(I)-Mediated Direct Arylation of 2′-Deoxyadenosines: Mechanistic Role of Cu(I) and Reactivity Comparisons with Related Purine Nucleosides

Pd/Cu-mediated direct arylation of 2′-deoxyadenosine with various aryl iodides provides 8-arylated 2′-deoxyadenosine derivatives in good yields. Following significant reaction optimization, it has been determined that a substoichiometric quantity of piperidine (secondary amine) in combination with cesium carbonate is necessary for effective direct arylation. The general synthetic protocol allows lower temperature direct arylations, which minimizes deglycosylation. The origin of the piperidine effect primarily derives from the in situ generation of Pd(OAc)2[(CH2)5NH]2. Various copper(I) salts have been evaluated; only CuI provides good yields of the 8-arylated-2′-deoxyadenosines. Copper(I) appears to have a high binding affinity for 2′-deoxyadenosine, which explains the mandatory requirement for stoichiometric amounts of this key component. The conditions are compared with more general direct arylation protocols, e.g., catalytic Pd, ligand, acid additives, which do not employ copper(I). In each case, no detectable arylation of 2′-deoxyadenosine was noted. The conformational preferences of the 8-aryl-2′-deoxyadenosine products have been determined by detailed spectroscopic (NMR) and single crystal X-ray diffraction studies. Almost exclusively, the preferred solution-state conformation was determined to be syn-C2′-endo (ca. 80%). The presence of a 2-pyridyl group at the 8-position further biases the solution-state equilibrium toward this conformer (ca. 88%), due to an additional H-bond between H1′ and the pyridyl nitrogen atom. The Pd/Cu catalyst system has been found to be unique for adenosine type substrates, the reactivity of which has been placed into context with the reported direct arylations of related 1H-imidazoles. The reactivity of other purine nucleosides has been assessed, which has revealed that both 2′-deoxyguanosine and guanosine are incompatible with the Pd/Cu-direct arylation conditions. Both substrates appear to hinder catalysis, akin to the established inhibitory effects in Suzuki cross-couplings with arylboronic acids

Pd(0)/Cu(I)-Mediated Direct Arylation of 2′-Deoxyadenosines: Mechanistic Role of Cu(I) and Reactivity Comparisons with Related Purine Nucleosides

Pd/Cu-mediated direct arylation of 2′-deoxyadenosine with various aryl iodides provides 8-arylated 2′-deoxyadenosine derivatives in good yields. Following significant reaction optimization, it has been determined that a substoichiometric quantity of piperidine (secondary amine) in combination with cesium carbonate is necessary for effective direct arylation. The general synthetic protocol allows lower temperature direct arylations, which minimizes deglycosylation. The origin of the piperidine effect primarily derives from the in situ generation of Pd(OAc)2[(CH2)5NH]2. Various copper(I) salts have been evaluated; only CuI provides good yields of the 8-arylated-2′-deoxyadenosines. Copper(I) appears to have a high binding affinity for 2′-deoxyadenosine, which explains the mandatory requirement for stoichiometric amounts of this key component. The conditions are compared with more general direct arylation protocols, e.g., catalytic Pd, ligand, acid additives, which do not employ copper(I). In each case, no detectable arylation of 2′-deoxyadenosine was noted. The conformational preferences of the 8-aryl-2′-deoxyadenosine products have been determined by detailed spectroscopic (NMR) and single crystal X-ray diffraction studies. Almost exclusively, the preferred solution-state conformation was determined to be syn-C2′-endo (ca. 80%). The presence of a 2-pyridyl group at the 8-position further biases the solution-state equilibrium toward this conformer (ca. 88%), due to an additional H-bond between H1′ and the pyridyl nitrogen atom. The Pd/Cu catalyst system has been found to be unique for adenosine type substrates, the reactivity of which has been placed into context with the reported direct arylations of related 1H-imidazoles. The reactivity of other purine nucleosides has been assessed, which has revealed that both 2′-deoxyguanosine and guanosine are incompatible with the Pd/Cu-direct arylation conditions. Both substrates appear to hinder catalysis, akin to the established inhibitory effects in Suzuki cross-couplings with arylboronic acids