nucleosomal dna  (Thermo Fisher)

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet:

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet: The BRD4 tandem bromodomains stably associate with H4 poly-acetylated nucleosomes in biolayer interferometry. ( A-D ) Biolayer interferometry binding assays of ( A ) BD1-2 with H4 poly-acetylated nucleosomes ([H4K5acK8acK12acK16ac] 2 ), ( B ) BD1-2 with unacetylated nucleosomes, ( C ) BD1 alone with H4 poly-acetylated nucleosomes, and ( D ) BD1 alone with unacetylated nucleosomes. Protein concentrations are indicated at the right of each plot. Note the difference in y-axis scale between ( A ) 1 to -0.5 nm and ( B-D ) 0.3 to -0.3 nm. Octet fitting of BD1-2 binding curves to H4 poly-acetylated nucleosomes (12.5, 6.25, and 3.12 μM) using a 2:1 heterogeneous ligand model produced R 2 values >0.98 (Fig. S2). Experiments were performed in triplicate.

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Stable Transfection, Binding Assay, Produced

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet: AlphaFold model suggesting a plausible mode of BRD4-S engagement with an H4 K12 acetylated nucleosome ([H4K12ac] 2 ). ( A ) Top-ranked AlphaFold model prediction of BRD4-S (blue) in complex with an H4K12ac nucleosome (H2A, golden; H2B, tomato; H3, green; H4, violet, Widom 601 DNA, grey). BD1 and BD2 are shaded in darker blue for clarity. ( B ) Rotated (45°) view of the modeled BRD4-S-H4K12ac nucleosome complex. ( C ) Zoom-in of the BD1 acetyl-lysine binding pocket highlighting residues Y97 and N140 positioned near H4K12ac. ( D ) Zoom-in of the BD2 acetyl-lysine binding pocket highlighting residues Y390 and N433 positioned near H4K12ac. This view also shows the relevant placement of the C-terminal BRD4-S region including the BID, ET, and phosphoregulatory sites, in the model.

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet: Bromodomain binding pocket mutations in the BRD4-S disrupt breast cancer growth and migration phenotypes. ( A ) Cell growth assay following BRD4-S depletion and complementation with the indicated 3xFLAG-BRD4-S constructs (mean ± SEM; n≥5 independent experiments). Statistical analysis as described in Methods. ( B ) Transwell migration assay following BRD4-S depletion and complementation with the indicated 3xFLAG-BRD4-S constructs (mean ± SEM; n ≥ 3 independent experiments). Statistical significance was calculated by two-tailed t-tests (***p<0.0005, ****p<0.0001). ( C ) Western blots of endogenous BRD4-S depletion and rescue by 3xFLAG-tagged BRD4-S (1-719) constructs in doxycycline-treated MDA-MB-231 cells. Blots were probed with an antibody raised for the N-terminus of BRD4 (BRD4-S; for endogenous ∼100 kDa), M2 FLAG (FLAG), and β-actin (Actin) ( D ) Graphical illustration of putative model for BRD4-S chromatin engagement and regulation of cancer cell growth and migration (BioRender, https://BioRender.com/7dfa8yg ). This model shows multiple possible binding modes of BRD4-S on poly-acetylated nucleosomes that would be difficult to distinguish or determine in biochemical and structural studies, yet could all contribute to recruitment of regulatory complexes and transcriptional control of nearby genes including those for cancer growth and migration.

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Binding Assay, Migration, Growth Assay, Construct, Transwell Migration Assay, Two Tailed Test, Western Blot, Control

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Schematic of approach to design, express, and characterize a panel of immunologically-silenced mAbs against cfDNA components (dsDNA, mononucleosomes, histones). Priming agent mAbs were generated using VH and VL amino acid sequences of mAbs that bind cfDNA components fused to a murine IgG2a backbone with L234A/L235A/P329G silencing mutations. B) EMSA of mAbs binding to free dsDNA or MNs, and quantification of binding interaction with biolayer interferometry. Free dsDNA binding was determined by the presence of shifted bands in the DNA lane. MN-only binding was determined by shift or disappearance of the MN band in the MN lane and no evidence of a shifted band in the DNA lane. For mAbs that bound free dsDNA or MN, the binding interaction was subsequently quantified with BLI. C) Measurement of binding kinetics (k on and k off ) and avidity (apparent K d ) to free dsDNA (left) and MN (right) of intact mAbs. D) Comparison of K d of dsDNA binders and MN binders to free dsDNA and MN. E) Interaction of MN binders with individual components of mononucleosomes and different dsDNA topologies, including individual histones, H2A/H2B dimer, H3/H4 tetramer, H2A/H2B/H3/H4 octamer, bent dsDNA (supercoiled pUC18), linear dsDNA (linearized pUC18, lin. pUC18), and intact MN (histone octamer + 147bp dsDNA). NB - no binding.

Article Snippet: Widom601 dsDNA, either free (cat. 16-0006, Epicypher) or histone bound in mononucleosomes (18-0005, Epicypher), was mixed to a final concentration of 0.2 ng/μL with each mAb binder to a concentration of 0.4 mg/mL in DPBS.

Techniques: Generated, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Degradation of free dsDNA and dsDNA in mononucleosomes in presence of 0.2U/mL of DNase I and mAb priming agents. IgG2a is an unrelated control antibody. Each dot represents the mean of two replicates. B) Experimental approach for testing impact of priming agents on clearance of free dsDNA and MN. C) Percentage of W601 DNA remaining in plasma 1 hour after injection of W601 either in free dsDNA form (left) or as MN (right), with or without dsDNA binding and MN binding priming agents. D) Percentage of cfDNA isolated from mouse plasma by various priming agents via immunoprecipitation using mAb-coupled magnetic beads, adjusted for background binding to beads alone. E) Percentage of cfDNA, W601 free dsDNA, W601 MN, and W601 MN with mild DNase I treatment isolated from mouse plasma using each MN-only mAb priming agent. F) cfDNA fragment length distribution in mice (n=8) with dashed lines at 167bp and 147bp (left) and percent of fragments <=147 bp and <=167 bp (right). * p < 0.05, ** p < 0.01, *** p < 0.001, ns - not significant.

Article Snippet: Widom601 dsDNA, either free (cat. 16-0006, Epicypher) or histone bound in mononucleosomes (18-0005, Epicypher), was mixed to a final concentration of 0.2 ng/μL with each mAb binder to a concentration of 0.4 mg/mL in DPBS.

Techniques: Control, Clinical Proteomics, Injection, Binding Assay, Isolation, Immunoprecipitation, Magnetic Beads

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: Antibody-based priming agents bind cfDNA in the bloodstream and protect it from clearance, enabling more to be collected in a subsequent blood draw. This study identified the key molecular determinants of priming activity. The optimal target binder was dsDNA, rather than mononucleosomes, and the priming activity was correlated with strength of binding to dsDNA, with the best priming agents having K d dsDNA < 10nM. The best dsDNA-binding priming agents had different magnitudes of DNase protection ability and impact on cfDNA fragmentation. In particular, one agent, DNA1, best preserved the endogenous fragmentation profile in cfDNA and protected short, informative cfDNA fragments at transcription factor binding sites from clearance. The Fc domain was found to be dispensable for the priming effect, suggesting that agents with more rapid clearance can still elicit a priming effect. Finally, we leveraged some of the principles identified in this study to engineer new single chain molecules that can similarly elicit a priming effect in tumor bearing mice, extending the space of priming agents to non-immunoglobulin dsDNA binding domains.

Article Snippet: Widom601 dsDNA, either free (cat. 16-0006, Epicypher) or histone bound in mononucleosomes (18-0005, Epicypher), was mixed to a final concentration of 0.2 ng/μL with each mAb binder to a concentration of 0.4 mg/mL in DPBS.

Techniques: Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Schematic of approach to design, express, and characterize a panel of immunologically-silenced mAbs against cfDNA components (dsDNA, mononucleosomes, histones). Priming agent mAbs were generated using VH and VL amino acid sequences of mAbs that bind cfDNA components fused to a murine IgG2a backbone with L234A/L235A/P329G silencing mutations. B) EMSA of mAbs binding to free dsDNA or MNs, and quantification of binding interaction with biolayer interferometry. Free dsDNA binding was determined by the presence of shifted bands in the DNA lane. MN-only binding was determined by shift or disappearance of the MN band in the MN lane and no evidence of a shifted band in the DNA lane. For mAbs that bound free dsDNA or MN, the binding interaction was subsequently quantified with BLI. C) Measurement of binding kinetics (k on and k off ) and avidity (apparent K d ) to free dsDNA (left) and MN (right) of intact mAbs. D) Comparison of K d of dsDNA binders and MN binders to free dsDNA and MN. E) Interaction of MN binders with individual components of mononucleosomes and different dsDNA topologies, including individual histones, H2A/H2B dimer, H3/H4 tetramer, H2A/H2B/H3/H4 octamer, bent dsDNA (supercoiled pUC18), linear dsDNA (linearized pUC18, lin. pUC18), and intact MN (histone octamer + 147bp dsDNA). NB - no binding.

Article Snippet: Streptavidin biosensor tips (cat. 18-5019, Sartorius) were used to immobilize mononucleosomes (16-0006, Epicypher), free dsDNA (18-0005, Epicypher) with the Widom601 sequence and a biotin tag, or biotinylated supercoiled or linearized pUC18 plasmid DNA, diluted to a final concentration of 8nM in kinetics buffer (5mg/ml BSA, 0.05% Tween-20 in PBS).

Techniques: Generated, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Degradation of free dsDNA and dsDNA in mononucleosomes in presence of 0.2U/mL of DNase I and mAb priming agents. IgG2a is an unrelated control antibody. Each dot represents the mean of two replicates. B) Experimental approach for testing impact of priming agents on clearance of free dsDNA and MN. C) Percentage of W601 DNA remaining in plasma 1 hour after injection of W601 either in free dsDNA form (left) or as MN (right), with or without dsDNA binding and MN binding priming agents. D) Percentage of cfDNA isolated from mouse plasma by various priming agents via immunoprecipitation using mAb-coupled magnetic beads, adjusted for background binding to beads alone. E) Percentage of cfDNA, W601 free dsDNA, W601 MN, and W601 MN with mild DNase I treatment isolated from mouse plasma using each MN-only mAb priming agent. F) cfDNA fragment length distribution in mice (n=8) with dashed lines at 167bp and 147bp (left) and percent of fragments <=147 bp and <=167 bp (right). * p < 0.05, ** p < 0.01, *** p < 0.001, ns - not significant.

Article Snippet: Streptavidin biosensor tips (cat. 18-5019, Sartorius) were used to immobilize mononucleosomes (16-0006, Epicypher), free dsDNA (18-0005, Epicypher) with the Widom601 sequence and a biotin tag, or biotinylated supercoiled or linearized pUC18 plasmid DNA, diluted to a final concentration of 8nM in kinetics buffer (5mg/ml BSA, 0.05% Tween-20 in PBS).

Techniques: Control, Clinical Proteomics, Injection, Binding Assay, Isolation, Immunoprecipitation, Magnetic Beads

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Design of priming agents using the 7 kDa dsDNA binding protein sso7d. Mono-valent, bi-valent, and tetra-valent constructs were designed using flexible short (G4S) 3 and long (G4S) 5 linkers fused to murine IgG2a CH2-CH3 domain carrying the LALAPG silencing domain, with addition on knob-in-hole mutations to promote heterodimerization for the mono-sso7d construct. B) Interferometry recordings of the interaction of sso7d priming agents with free dsDNA (“dsDNA binding”) and MN (“MN binding”). For binding to dsDNA, the range of priming agent concentrations tested was 1.56-50nM, whereas for binding to MN, the range of concentrations tested was 12.5-400nM in order to capture weaker interactions. C) Estimated avidity (K d ) for binding to dsDNA and MN for sso7d priming agents compared to aST3. D) Experimental approach for testing the effect of sso7d priming agents on cfDNA and ctDNA recovery. E) Fold-change in plasma cfDNA concentration after administration of each priming agent, based on qPCR quantification. F) Fold-change in number of ctDNA molecules detected in plasma after administration of priming agents. G) Priming effect versus K d to dsDNA and MN for sso7d priming agents. * p < 0.05, ** p < 0.01, *** p < 0.001, ns - not significant. Points represent mean and intervals represent 95% confidence intervals in G. Box plots represent median and interquartile range.

Article Snippet: Streptavidin biosensor tips (cat. 18-5019, Sartorius) were used to immobilize mononucleosomes (16-0006, Epicypher), free dsDNA (18-0005, Epicypher) with the Widom601 sequence and a biotin tag, or biotinylated supercoiled or linearized pUC18 plasmid DNA, diluted to a final concentration of 8nM in kinetics buffer (5mg/ml BSA, 0.05% Tween-20 in PBS).

Techniques: Binding Assay, Construct, Clinical Proteomics, Concentration Assay

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: Antibody-based priming agents bind cfDNA in the bloodstream and protect it from clearance, enabling more to be collected in a subsequent blood draw. This study identified the key molecular determinants of priming activity. The optimal target binder was dsDNA, rather than mononucleosomes, and the priming activity was correlated with strength of binding to dsDNA, with the best priming agents having K d dsDNA < 10nM. The best dsDNA-binding priming agents had different magnitudes of DNase protection ability and impact on cfDNA fragmentation. In particular, one agent, DNA1, best preserved the endogenous fragmentation profile in cfDNA and protected short, informative cfDNA fragments at transcription factor binding sites from clearance. The Fc domain was found to be dispensable for the priming effect, suggesting that agents with more rapid clearance can still elicit a priming effect. Finally, we leveraged some of the principles identified in this study to engineer new single chain molecules that can similarly elicit a priming effect in tumor bearing mice, extending the space of priming agents to non-immunoglobulin dsDNA binding domains.

Article Snippet: Streptavidin biosensor tips (cat. 18-5019, Sartorius) were used to immobilize mononucleosomes (16-0006, Epicypher), free dsDNA (18-0005, Epicypher) with the Widom601 sequence and a biotin tag, or biotinylated supercoiled or linearized pUC18 plasmid DNA, diluted to a final concentration of 8nM in kinetics buffer (5mg/ml BSA, 0.05% Tween-20 in PBS).

Techniques: Activity Assay, Binding Assay