GAE4 Antibody

What is BG4 antibody and what are its primary applications in G-quadruplex research?

BG4 is a monoclonal single chain antibody generated by phage display with high affinity and specificity for G-quadruplex (G4) DNA structures. It was developed specifically to investigate the presence and distribution of G4 structures in the genome. BG4 serves as a valuable tool for visualizing G4 structures in fixed cells through immunofluorescence and for identifying G4-containing genomic regions via techniques like ChIP-seq. The antibody has been instrumental in providing direct evidence for the existence of G4 structures within cells, which was previously a subject of debate in the scientific community .

The primary applications of BG4 include:

• Immunostaining to detect and quantify G4 structures within cell nuclei

• Chromatin immunoprecipitation to identify G4-rich regions in the genome

• In vitro binding studies to characterize G4-forming sequences

• Investigating the relationship between G4 structures and cellular processes

How is BG4 prepared and purified for research applications?

BG4 antibody can be overexpressed in bacterial systems and purified using affinity column chromatography. The purification process typically employs a His-tag system with imidazole elution. According to reported protocols, BG4 is most effectively recovered in the 800 mM to 1 M imidazole fractions. After collection, these fractions should be pooled, dialyzed against an appropriate buffer, and stored under conditions that maintain antibody activity .

The quality and identity of purified BG4 can be confirmed through:

• Denaturing PAGE to assess purity

• Western blotting with appropriate anti-tag antibodies

• Functional assays demonstrating binding to known G4-forming oligonucleotides

Researchers should verify that purified BG4 batches retain specific binding activity to G4 DNA (such as oligomers derived from the Hif1α gene) while showing no binding to complementary C-rich sequences .

What is the binding affinity of BG4 for G-quadruplex DNA?

BG4 demonstrates robust binding affinity for G4 DNA structures. Biolayer interferometry (BLI) studies have revealed a dissociation constant (Kd) of 17.4 ± 0.588 nM (mean ± SD) for G4 DNA derived from the Hif1α gene. Previous reports have indicated an even higher affinity with a Kd of approximately 2.0 nM .

The binding kinetics can be measured using techniques such as:

• Biolayer interferometry with 5′-biotinylated G4 oligomers immobilized on streptavidin sensor surfaces

• Electrophoretic mobility shift assays (EMSA) with increasing concentrations of BG4

• Competition assays using labeled and unlabeled G4 substrates

These quantitative measurements confirm that BG4 binds to G4 structures with high affinity, making it suitable for both in vitro and cellular detection applications .

What structural features of G-quadruplexes are recognized by BG4?

BG4 demonstrates distinct structural preferences in G4 recognition. Through comprehensive binding studies using electrophoretic mobility shift assays (EMSA) and circular dichroism (CD) spectroscopy, researchers have established that BG4 primarily recognizes parallel G-quadruplex conformations. The antibody shows effective binding to both intermolecular and intramolecular G4 structures, but only when they adopt a parallel orientation .

Notably, BG4 fails to bind efficiently to:

• Antiparallel G-quadruplex structures, including those derived from human telomeric regions (SV23, SV25, SV27)

• Hybrid G-quadruplex conformations (tested with oligomers SMJ50-61)

• Complementary C-rich sequences (KD53, MN89, SMJ36)

This conformational specificity is crucial for experimental design and interpretation of results. The antibody can bind to various G4 structures with GNG sequences (KD49, KD81, KD83), with some exceptions (KD85), suggesting that certain sequence variations within G4 motifs may affect recognition .

How can researchers validate the specificity of BG4 binding to G4 DNA?

To ensure the specificity of BG4 binding to G4 DNA, researchers should implement multiple validation strategies:

• Competition assays: Incubate a fixed concentration of BG4 with radiolabeled G4 DNA (e.g., RT17) and compete with increasing concentrations of either unlabeled G4 DNA or random sequences. Specific binding should show decreased complex formation only with G4 competitor but not with random sequences .

• Control antibodies: Test binding using various non-G4-specific antibodies (e.g., antibodies against AID, GFP, PAX5, MBP, GAPDH, IgG). Lack of binding with these antibodies confirms BG4 specificity .

• Structural variants: Compare BG4 binding across different G4 structural conformations using CD spectroscopy to confirm structural preferences .

• Substrate titrations: Perform dose-response experiments with increasing concentrations of BG4 against fixed amounts of G4 DNA and control sequences .

These rigorous validation approaches ensure that experimental observations with BG4 truly reflect G4 structure presence rather than non-specific interactions.

What are the limitations of using BG4 for G-quadruplex detection in cellular contexts?

While BG4 is a powerful tool for G4 detection, researchers should be aware of several important limitations:

• Structural selectivity: BG4 predominantly recognizes parallel G4 conformations, potentially underrepresenting antiparallel and hybrid structures that may exist in the genome. This selective recognition means that BG4-based studies may not provide a complete picture of all G4 structures present in cells .

• Accessibility constraints: In cellular immunostaining applications, BG4 access may be limited by chromatin compaction, protein binding, or other nuclear factors that could mask G4 structures, leading to potential false negatives.

• Fixation artifacts: Cell fixation methods required for immunostaining may alter native G4 conformations or induce artificial G4 formation.

• Binding perturbation: BG4 binding itself may stabilize or alter G4 structures, potentially affecting their natural dynamics and interactions with cellular proteins.

• Resolution limitations: Standard immunofluorescence techniques may not distinguish between closely spaced G4 structures, limiting quantitative analysis of G4 abundance.

Researchers should consider these limitations when designing experiments and interpreting results from BG4-based G4 detection studies.

How does cell cycle phase influence BG4 foci formation and what are the implications?

Immunostaining with BG4 in human cells has revealed that G-quadruplex structures are present throughout all cell cycle phases, with the maximum number of foci observed during S phase. This observation suggests a replication-dependent formation of G4 structures, which aligns with the understanding that DNA unwinding during replication provides opportunities for non-canonical DNA structure formation .

The cell cycle-dependent variations in BG4 foci have several important implications:

• DNA replication vulnerability: The increased formation of G4 structures during S phase suggests these regions may be particularly vulnerable to replication stress and genomic instability.

• Regulatory mechanisms: The dynamics of G4 formation across the cell cycle may represent a regulatory mechanism for controlling gene expression in a cell cycle-dependent manner.

• Helicase activity correlation: The modulation of BG4 foci upon knockdown of G4-resolving helicases like WRN suggests active regulation of G4 structures during cell cycle progression .

• Experimental timing considerations: Researchers investigating G4 structures should consider cell synchronization or cell cycle analysis in their experimental designs to account for these variations.

This cell cycle dependence provides a framework for designing experiments that investigate the functional roles of G4 structures in processes like DNA replication, transcription, and genome stability.

How can BG4 be used in combination with other techniques to study G-quadruplex biology?

BG4 antibody can be integrated into multi-dimensional research approaches to provide comprehensive insights into G-quadruplex biology:

• BG4 with G4 ligands: Treating cells with G4-stabilizing compounds (e.g., pyridostatin) before BG4 immunostaining increases the number of detectable foci, allowing for assessment of G4 stabilization effects and potential therapeutic applications .

• BG4 ChIP-seq: Chromatin immunoprecipitation with BG4 followed by next-generation sequencing has identified over 10,000 G4 structures in regulatory, nucleosome-depleted regions of human chromatin, providing genome-wide maps of G4 distribution .

• BG4 with helicase knockdowns: Modulating the expression of G4-resolving helicases (such as WRN) while monitoring BG4 foci can reveal mechanisms of G4 regulation and metabolism in cells .

• BG4 with live-cell G4 probes: Correlating BG4 immunostaining with data from fluorescent G4 probes (like SiR-PyPDS) that work in live cells can validate observations across different detection methods .

• BG4 with transcriptomics: Combining BG4 mapping with RNA-seq data can identify correlations between G4 formation and gene expression patterns.

This integrative approach maximizes the utility of BG4 antibody while compensating for its limitations through complementary techniques.

What controls should be included when using BG4 for immunofluorescence studies?

When conducting immunofluorescence studies with BG4 antibody, several critical controls should be implemented to ensure reliable and interpretable results:

• Antibody specificity controls:

  • Secondary antibody-only control: Cells treated with FLAG antibody (to detect the FLAG tag on BG4) and secondary antibody without prior BG4 incubation should show no foci formation .

  • Irrelevant primary antibody control: Using an unrelated antibody of the same isotype to verify that foci formation is specific to BG4.

• Biological controls:

  • G4 helicase knockdown: Cells with reduced expression of G4-resolving helicases like WRN should show altered BG4 foci patterns, confirming biological relevance .

  • Cell cycle synchronization: Comparing BG4 staining patterns across synchronized cell populations to verify cell cycle-dependent changes.

• Technical controls:

  • Cell fixation controls: Different fixation methods should be compared to ensure they don't artificially induce or disrupt G4 structures.

  • Multiple cell lines: Testing BG4 staining across different cell types (e.g., REH, Nalm6, HEK293T, HeLa) provides insight into the consistency of G4 formation across cellular contexts .

• Signal validation controls:

  • DNase treatment: Pre-treating fixed cells with DNase should eliminate BG4 staining, confirming DNA-dependent binding.

  • G4-stabilizing compounds: Treatment with known G4 ligands should increase BG4 foci, serving as a positive control.

Proper implementation of these controls ensures that observed BG4 foci truly represent G-quadruplex structures rather than artifacts or non-specific binding.

What are the optimal experimental conditions for BG4 binding assays?

For optimal BG4 binding assays, researchers should consider the following experimental parameters:

• Protein concentration: BG4 shows concentration-dependent binding to G4 DNA, with detectable binding starting from 150 ng and improving with increasing concentration (up to 1.3 μg tested) .

• Substrate preparation:

  • G4 structures should be properly folded in appropriate buffers containing potassium ions

  • For in vitro studies, oligonucleotides derived from known G4-forming sequences (e.g., Hif1α) serve as positive controls

  • Complementary C-rich sequences provide essential negative controls

• Binding reaction conditions:

  • Buffer composition should maintain G4 stability while supporting antibody binding

  • Incubation temperature and time should be optimized (typically room temperature for 30-60 minutes)

  • For EMSA, gel conditions should be non-denaturing to preserve G4 structures and antibody-DNA complexes

• Detection methods:

  • For EMSA with radiolabeled substrates, 20 nM oligonucleotide concentration has been effective

  • For BLI studies, 5′-biotinylated oligomers immobilized on streptavidin sensors with BG4 at 50-320 nM range provide reliable binding curves

  • For immunofluorescence, a sequential detection system using anti-FLAG primary antibody followed by fluorophore-conjugated secondary antibody yields optimal signal-to-noise ratio

Careful optimization of these conditions ensures reproducible and meaningful results in BG4 binding experiments.

How can BG4 be used to investigate G-quadruplex structures in telomeric regions?

BG4 antibody offers several methodological approaches to study G-quadruplex structures within telomeric regions:

• Plasmid-based assays: BG4 can bind to G4-DNA within telomere sequences in supercoiled plasmids, allowing for investigation of telomeric G4 formation under topological constraints that mimic native chromosomes .

• Immunofluorescence co-localization: BG4 immunostaining can be combined with telomere-specific FISH probes to quantify the proportion of telomeres containing G4 structures across different cell types or conditions.

• Telomere dysfunction models: BG4 staining patterns can be analyzed in cells with telomere maintenance defects (e.g., telomerase-negative cells, shelterin protein knockdowns) to assess correlations between G4 formation and telomere integrity.

• Telomere-specific ChIP: BG4 chromatin immunoprecipitation followed by telomere-specific qPCR can provide quantitative measures of G4 abundance at telomeres across different cellular contexts.

When designing telomeric G4 studies with BG4, researchers should note that the antibody appears to recognize G4 structures in telomeric sequences when they adopt a parallel conformation. The antibody may not effectively detect antiparallel G4 structures that can form with certain telomeric repeats, as demonstrated by the lack of binding to antiparallel G4 structures derived from human telomeric regions (SV23, SV25, SV27) .

How can researchers differentiate between false positives and true G-quadruplex structures when using BG4?

Distinguishing true G-quadruplex structures from false positives when using BG4 requires a multi-faceted validation approach:

• Sequence verification:

  • G4 structures typically form in G-rich sequences with the motif G₃₊N₁₋₇G₃₊N₁₋₇G₃₊N₁₋₇G₃₊

  • Regions with BG4 binding should be analyzed for the presence of canonical G4 motifs

  • Mere presence of G4 motifs in duplex DNA is insufficient for antibody recognition, emphasizing the importance of structural verification

• Structural confirmation:

  • Circular dichroism (CD) spectroscopy can confirm G4 formation in vitro

  • Parallel G4 structures (which BG4 preferentially binds) show characteristic positive peaks at ~260 nm and negative peaks at ~240 nm in CD spectra

  • Oligonucleotides with confirmed G4 formation by CD should be used as positive controls

• Competition experiments:

  • Competition with known G4 ligands can help validate specific BG4 binding

  • Unlabeled G4 DNA should compete effectively with labeled G4 for BG4 binding

  • Random sequences should not compete for BG4 binding

• Biological validation:

  • G4-resolving helicase (WRN) knockdown should increase BG4 foci

  • G4-stabilizing compounds should increase BG4 signal

  • G4 formation should correlate with biological processes (e.g., transcription, replication) known to involve G4 structures

By implementing these rigorous validation approaches, researchers can minimize false positives and ensure that BG4 signals accurately represent G-quadruplex structures in their experimental systems.

What emerging applications of BG4 antibody are advancing G-quadruplex research?

Several innovative applications of BG4 antibody are pushing the boundaries of G-quadruplex research:

• G4 ChIP-seq: Chromatin immunoprecipitation with BG4 followed by next-generation sequencing has identified over 10,000 G4 structures in regulatory, nucleosome-depleted regions of human chromatin. This genome-wide mapping approach continues to evolve with improved resolution and sensitivity .

• Single-cell G4 analysis: Adapting BG4 immunofluorescence for single-cell analysis could reveal cell-to-cell variations in G4 formation, potentially uncovering stochastic aspects of G4 biology.

• Super-resolution microscopy: Combining BG4 with techniques like STORM or PALM could provide nanoscale resolution of G4 distribution within nuclei, potentially revealing spatial relationships between G4 structures and nuclear compartments.

• G4 dynamics studies: Using BG4 in combination with live-cell G4 probes like SiR-PyPDS allows researchers to correlate fixed-cell observations with dynamic G4 behaviors in living cells .

• Tissue-specific G4 mapping: Applying BG4 immunohistochemistry to tissue sections could reveal tissue-specific patterns of G4 formation with potential clinical implications.

These emerging applications demonstrate the versatility of BG4 as a research tool and highlight its continued importance in advancing our understanding of G-quadruplex biology.

How might G-quadruplex research using BG4 antibody impact therapeutic development?

G-quadruplex research using BG4 antibody has several potential implications for therapeutic development:

• Target identification: BG4 ChIP-seq and immunofluorescence studies can identify genomic regions and cellular contexts with abundant G4 structures, potentially revealing new therapeutic targets.

• Drug mechanism studies: BG4 can be used to confirm the G4-targeting activity of small molecules in cells, providing mechanistic insights into drug candidates that stabilize G4 structures.

• Biomarker development: Patterns of BG4 binding in patient-derived samples might serve as biomarkers for diseases associated with aberrant G4 formation or resolution.

• Resistance mechanisms: BG4 studies in treatment-resistant cells could reveal alterations in G4 biology that contribute to therapeutic resistance.

• Cell cycle-specific targeting: The observation that G4 structures are most abundant during S phase, as revealed by BG4 immunostaining, suggests potential for cell cycle-specific therapeutic strategies targeting cells with elevated G4 formation .

• Helicase inhibition effects: The increase in BG4 foci upon knockdown of G4-resolvase WRN indicates that targeting G4-resolving helicases could be a viable therapeutic approach, with BG4 providing a readout for efficacy .

By advancing our fundamental understanding of G-quadruplex biology, BG4-based research is laying the groundwork for novel therapeutic approaches targeting these non-canonical DNA structures.