N3-kethoxal: Membrane-Permeable Azide Probe for RNA and DNA Accessibility Mapping

Executive Summary: N3-kethoxal (CAS 2382756-48-9) is a synthetic nucleic acid probe that selectively labels unpaired guanine bases in RNA and single-stranded DNA, introducing an azide for click chemistry-based enrichment [product]. It is membrane-permeable, enabling applications in live cells and in vitro assays [internal]. Benchmarking studies validate its use in KAS-seq and KAS-ATAC workflows to map accessible chromatin and ssDNA regions genome-wide (Marinov & Greenleaf 2025). N3-kethoxal's high solubility and chemical specificity underpin robust labeling and low background. It is pivotal for probing RNA secondary structure, mapping active regulatory DNA elements, and studying nucleic acid interactions.

Biological Rationale

Gene regulation depends on the dynamic accessibility of nucleic acids. Cis-regulatory elements (cREs) such as promoters and enhancers are typically nucleosome-depleted when active, exposing single-stranded DNA (ssDNA) regions during transcription initiation and elongation (Marinov & Greenleaf 2025). RNA molecules frequently form secondary and tertiary structures, with unpaired guanines marking flexible or functionally relevant regions. Traditional approaches like DNase I hypersensitivity and ATAC-seq profile chromatin accessibility but lack direct detection of ssDNA or RNA structure. N3-kethoxal overcomes these limitations by covalently tagging unpaired guanines, offering direct, high-specificity readouts of nucleic acid accessibility and conformation (see extended analysis: mechanistic precision). This enables comprehensive mapping of regulatory networks and nucleic acid interaction landscapes in both genomic DNA and RNA pools.

Mechanism of Action of N3-kethoxal

N3-kethoxal is a membrane-permeable, azide-functionalized compound with the chemical formula C6H11N3O4 and molecular weight 189.17 g/mol [A8793 product page]. Upon exposure to nucleic acids, N3-kethoxal reacts specifically with the N1 and N2 positions of unpaired guanine residues in ssDNA or exposed RNA loops. This forms a stable covalent adduct, introducing an azide group. The azide functionality enables bioorthogonal labeling through click chemistry, such as copper-catalyzed azide-alkyne cycloaddition (CuAAC), for downstream enrichment or visualization (see: biotin-azide workflows).

Key chemical features of N3-kethoxal include:

• High solubility: ≥94.6 mg/mL in DMSO, ≥24.6 mg/mL in water, ≥30.4 mg/mL in ethanol (room temperature).
• 98.00% purity, ensuring low background labeling.
• Membrane permeability, enabling live-cell and fixed-cell labeling.
• Stability: Store at -20°C; avoid long-term storage in solution.

The reaction is rapid (minutes scale) and highly selective for unpaired (not Watson-Crick paired) guanines, minimizing off-target effects in double-stranded DNA or structured RNA regions.

Evidence & Benchmarks

• N3-kethoxal enables covalent and selective labeling of unpaired guanine bases in both RNA and ssDNA under physiological conditions (Marinov & Greenleaf 2025, DOI).
• KAS-ATAC sequencing, which utilizes N3-kethoxal labeling, captures genomic DNA fragments that are simultaneously accessible and contain ssDNA bubbles (DOI).
• The labeled DNA can be efficiently conjugated to biotin using click chemistry for pulldown and library generation (DOI).
• KAS-seq and KAS-ATAC methods relying on N3-kethoxal show high sensitivity in genome-wide mapping of active transcriptional regulatory elements, outperforming traditional DNase and ATAC-seq in identifying ssDNA regions (DOI).
• RNA structure probing with N3-kethoxal is compatible with both in vitro refolded RNA and in vivo labeling protocols, with low cytotoxicity at working concentrations (internal).

Applications, Limits & Misconceptions

N3-kethoxal is suited for a range of nucleic acid research applications, including:

• RNA secondary and tertiary structure mapping: Identifies flexible, unpaired guanine residues in cellular or purified RNA.
• Genomic mapping of accessible DNA: Labels ssDNA bubbles at active promoters, enhancers, and transcriptionally engaged regions, as shown in KAS-ATAC and KAS-seq (DOI).
• RNA-protein proximity labeling: Enables detection of RNA-protein interactions via proximity-dependent labeling strategies.
• RNA-RNA interaction dynamics: Probes structural rearrangements and base accessibility in ribonucleoprotein complexes.
• Click chemistry compatibility: Azide handle allows downstream labeling, enrichment, and imaging using alkyne-modified reagents.

For additional optimized protocols and troubleshooting, see this guide, which this article updates with new data on genome-wide mapping and live-cell workflows.

Common Pitfalls or Misconceptions

• Not all guanine residues are labeled: Only unpaired or solvent-accessible guanines react; paired or protected guanines are not modified.
• Double-stranded DNA is largely inert: N3-kethoxal does not efficiently label dsDNA without transient base pair opening.
• Long-term solution storage reduces activity: N3-kethoxal should be stored dry at -20°C and freshly prepared for each use.
• Not suitable for methylation status detection: The probe detects ssDNA/RNA accessibility, not base modifications.
• High concentrations may affect cell viability: Use published working concentrations (typically <10 mM) to minimize cytotoxicity.

Workflow Integration & Parameters

N3-kethoxal is delivered as a liquid reagent for straightforward integration into nucleic acid labeling protocols. Standard workflows include:

1. Labeling: Incubate cells or purified RNA/DNA with N3-kethoxal at 37°C for 5–30 minutes in compatible buffer (e.g., PBS, pH 7.4).
2. Quenching: Remove excess reagent and quench with buffer containing guanosine or other nucleophile.
3. Click Chemistry: Perform copper-catalyzed azide-alkyne cycloaddition (CuAAC) with biotin-alkyne or fluorophore-alkyne for detection or enrichment.
4. Pulldown & Analysis: For DNA applications (e.g., KAS-ATAC), enrich labeled fragments with streptavidin beads, followed by library prep and sequencing (DOI).
5. Controls: Include no-probe and denatured nucleic acid controls to assess background and specificity.

Shipping is on Blue Ice for small molecules and Dry Ice for modified nucleotides; see the A8793 kit page for logistics and storage details.

Conclusion & Outlook

N3-kethoxal is a next-generation, azide-functionalized nucleic acid probe that uniquely enables direct, covalent labeling of unpaired guanines in both RNA and ssDNA. Its compatibility with live-cell and in vitro workflows, bioorthogonal click chemistry, and genome-wide mapping techniques positions it as a core tool for regulatory genomics and RNA structure research. Ongoing developments in multi-omic profiling and single-molecule analysis are likely to further extend its impact. For a broader perspective on translational and mechanistic advances, see the thought-leadership article here, which this article complements by focusing on workflow integration and benchmarking.