Journal: Nucleic Acids Research

Article Title: RSRC2 is a novel RNA-binding protein that safeguards mitotic fidelity by interacting with the lncRNA C1QTNF1-AS1

doi: 10.1093/nar/gkag229

Figure Lengend Snippet: C1QNTF1–AS1 interacts with RSRC2, an RBP of unknown function. ( A ) Schematic representation of the C1QTNF1–AS1 and C1QTNF1/CTRP1 genomic landscape ( C1QTNF1–AS1 annotated as NR_040 018/NR_040 019 in RefSeq; Gencode gene ENSG00000265096 ; chr17:79019209-79027601, hg38). ( B ) Maximum intensity projections of representative images of C1QTNF1–AS1 exon smRNA FISH in HCT116 showing its nuclear localization. Nuclei were stained with DAPI (magenta) and outlined with a dashed circle. Right panel: quantification of total transcript in the nucleus (N) and cytoplasm (C), solid line represents the mean. N = 3 ( n (HCT116) =398). ( C ) Expression levels of C1QTNF1–AS1 in HCT116 cells following depletion with three LNA gapmers targeting either exon 3 (LNA 1) or first intron of C1QTNF1–AS1 (LNA 2, 3), as measured by qPCR. Primers spanning mature (ex2-3) C1QTNF1–AS1 were used. Results are presented relative to negative control (Ctl A) LNA; N = 3. ( D ) Schematic representation of workflow for the ASO pulldown of C1QTNF1–AS1 in HCT116 cells. Five different ASOs targeting different regions of the C1QTNF1–AS1 locus were used, with luciferase (Luc) ASOs as a negative control. Pulldown efficacy was assessed by qPCR , and proteins were identified using LC-MS analysis (see Materials and methods). ( E ) The volcano plot highlighting proteins enriched in C1QTNF1–AS1 pulldown using ASO 1, 3, and 5 versus Luc. Significant C1QTNF1–AS1 protein interactors are highlighted in red (FDR 5%). ( F ) RIP-qPCR from HCT116 extracts. Left panel: Western blot of RSRC2 in the input and IP samples to show RSRC2 IP efficiency compared to IgG. Right panel: RIP-qPCR showing association of RSRC2 with C1QTNF1–AS1 transcript. GAPDH was used as negative control RNA for RSRC2 RIP. RIP enrichments are presented as % of input RNA (normalized to IgG); N = 3. ( G ) Relative expression of RSRC2 in HCT116 cells following siRNA-mediated depletion of RSRC2, as measured by qPCR. Results are presented relative to control siRNA (Ctl); N = 3. ( H ) Representative western blot showing RSRC2 protein expression in HCT116 cells following siRNA-mediated depletion of RSRC2. β-Tubulin and Ponceau staining were used as loading controls. An asterisk indicates an unspecific RSRC2 protein band. ( I ) Densitometric analysis of RSRC2 levels from panel (H) relative to control siRNA (Ctl); N = 3. ( J ) Interaction intensities between C1QTNF1–AS1 and the indicated proteins show that C1QTNF1–AS1 interacts with RSRC2. In-cell interactions were measured in an incPRINT experiment where MS2-tagged C1QTNF1–AS1 RNA was co-expressed with a set of FLAG-tagged proteins in HEK293T cells harbouring a luciferase detector fused to the MS2 coat protein (MS2CP). Upon the formation of FLAG–protein–RNA–MS2–MS2CP ternary complexes, RNA–protein interactions were measured by luciferase activity . eGFP (enhanced green fluorescent protein) was used as a negative control and PABPC3 (a polyadenylated RBP) was used to control for RNA expression. Xist(C) -MS2 vector was used alongside C1QTNF1AS1 –MS2 as a positive control for RNA–protein interactions. RLU are relative light unit; N = 4. ( K ) Protein expression levels were estimated from horseradish peroxidase ELISA of FLAG-tagged proteins in the same experiment as in panel (J). Error bars in all panels are shown as mean ± S.E.M.; scale bar: 5 μm; N = number of cells analysed. An unpaired t -test with Welch’s correction was applied in panels (C), (G), and (I). Unpaired t -test was used in panel (F). * <0.05, **<0.01, and ****<0.0001.

Article Snippet: C1QTNF1–AS1 insert was cloned upstream of the MS2 stem-loop sequence of the 10xMS2 vector using a Gibson assembly cloning kit (E5510S, NEB) at 50°C for 15 min.

Techniques: Staining, Expressing, Negative Control, Luciferase, Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Activity Assay, RNA Expression, Plasmid Preparation, Positive Control, Enzyme-linked Immunosorbent 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: 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