N3-kethoxal: Azide-Functionalized Probe for RNA and ssDNA Mapping

Executive Summary: N3-kethoxal is a synthetic, membrane-permeable nucleic acid probe that reacts specifically with unpaired guanines in RNA and single-stranded DNA, forming stable covalent adducts and introducing an azide group for click chemistry labeling (A8793 product page). This selectivity enables high-resolution mapping of nucleic acid structures, detection of accessible DNA regions, and profiling of RNA-protein or RNA-RNA interactions (Wang et al., 2024). N3-kethoxal exhibits high solubility in DMSO (≥94.6 mg/mL), water (≥24.6 mg/mL), and ethanol (≥30.4 mg/mL), with a molecular weight of 189.17 Da and purity ≥98%. Its robust performance in both in vitro and cellular workflows distinguishes it from less selective or membrane-impermeable alternatives (related review). Proper storage at -20°C is required to maintain stability.

Biological Rationale

Nucleic acid structure and accessibility govern key cellular processes such as transcription, translation, and genome maintenance (Wang et al., 2024). R-loops, which are RNA-DNA hybrid structures with a displaced single-stranded DNA, influence DNA replication, repair, and transcriptional regulation. Unpaired guanine bases in RNA and single-stranded DNA regions often serve as critical regulatory elements or intermediates in these processes. Chemical probes that can selectively label such unpaired nucleotides provide essential tools for mapping secondary and tertiary nucleic acid structures, elucidating RNA-protein proximity, and studying the dynamics of R-loops or CRISPR genome editing events. The specificity for unpaired guanine distinguishes N3-kethoxal from non-selective alkylators, allowing for atomic-level resolution in structure-function studies.

Mechanism of Action of N3-kethoxal

N3-kethoxal (3-(2-azidoethoxy)-1,1-dihydroxybutan-2-one) features an azide functional group and a reactive keto-aldehyde moiety. Upon cellular or in vitro application, the compound diffuses across membranes due to its small size and hydrophilicity. The dihydroxybutanone core reacts rapidly and covalently with the N1 and N2 positions of unpaired guanine bases in nucleic acids, forming stable adducts (A8793 datasheet). This reaction occurs preferentially in single-stranded or loop regions of RNA and DNA, leaving double-stranded or highly structured regions largely unmodified. The installed azide group enables subsequent bioorthogonal click chemistry, such as strain-promoted azide-alkyne cycloaddition (SPAAC), for fluorescent or affinity labeling. This modularity allows for downstream visualization, enrichment, or proteomic interrogation of labeled nucleic acids.

Evidence & Benchmarks

• N3-kethoxal covalently modifies unpaired guanine residues in RNA and single-stranded DNA, enabling single-nucleotide resolution mapping of accessible regions (Wang et al., 2024).
• The probe demonstrates high solubility in DMSO (≥94.6 mg/mL), water (≥24.6 mg/mL), and ethanol (≥30.4 mg/mL) at room temperature, supporting diverse experimental workflows (A8793 technical sheet).
• N3-kethoxal enables in vivo and in vitro RNA secondary structure probing, outperforming classical kethoxal in cell-permeability and click-compatibility (Pyronaridine Review).
• Adduct formation is stable under neutral to mildly basic buffer conditions (pH 7.0–8.0), with reaction times as short as 10–30 minutes at 25–37°C (A8793 datasheet).
• Unlike nonspecific alkylators, N3-kethoxal does not induce significant genome-wide DNA damage or cytotoxicity at working concentrations (Wang et al., 2024).

For a detailed comparison of single-molecule sensitivity and structural mapping, see the article 'N3-kethoxal: Pioneering Single-Molecule Mapping of RNA and DNA', which this article extends by including recent evidence on genomic stability and in vivo performance.

Applications, Limits & Misconceptions

Major Applications

• RNA Secondary Structure Probing: Enables nucleotide-resolution mapping of accessible guanines in living cells and purified RNA.
• Genomic Mapping of Accessible DNA: Facilitates detection of single-stranded or open regions, including R-loops and CRISPR off-targets.
• RNA-RNA and RNA-Protein Interaction Profiling: Permits proximity labeling and interactome studies via click chemistry conjugation.
• Click Chemistry Labeling: The azide handle supports modular conjugation for imaging, enrichment, or mass spectrometry.
• Single-Molecule and High-Throughput Workflows: Compatible with advanced sequencing and single-molecule platforms.

For a perspective on workflow streamlining and sensitivity advances, see 'N3-kethoxal: Precision Membrane-Permeable Nucleic Acid Probe', which this article clarifies by providing experimental boundary conditions.

Common Pitfalls or Misconceptions

• Does not label double-stranded or fully base-paired guanine: N3-kethoxal is selective for unpaired or looped guanines; it does not react with base-paired guanines in canonical helices (Wang et al., 2024).
• Not suitable for long-term storage in solution: The probe should be stored at -20°C as a solid to maintain reactivity, as solutions may degrade over time (A8793 datasheet).
• Not a general DNA damage inducer: At recommended concentrations, does not introduce widespread genotoxic lesions or affect cell viability (Wang et al., 2024).
• Azide group is inert without click partner: The introduced azide does not confer a detectable signal unless a compatible alkyne or reporter is applied.
• Not a methylation or alkylation marker: N3-kethoxal modifies guanine bases but does not report on naturally occurring methylation or alkylation events.

Workflow Integration & Parameters

N3-kethoxal is supplied as a high-purity (≥98%) liquid or solid and should be stored at -20°C. For labeling, prepare fresh working solutions in DMSO, water, or ethanol. Typical reaction conditions are 0.1–2 mM probe, 10–30 minutes incubation at 25–37°C, pH 7.0–8.0. For in vitro RNA or DNA labeling, apply to purified nucleic acids in buffered solution; for in vivo applications, add directly to cell culture media. After covalent modification, perform click chemistry (e.g., with DBCO- or alkyne-functionalized fluorophores or beads) to visualize or isolate labeled nucleic acids. Downstream processing may involve RNA-seq, ChIP-seq, or mass spectrometry. Shipping is on Blue Ice (small molecules) or Dry Ice (modified nucleotides). For workflow optimization and troubleshooting, see the extended discussion in 'N3-kethoxal: Next-Generation Probing for Dynamic Nucleic Acids', which this article updates by integrating latest solubility and stability parameters.

Conclusion & Outlook

N3-kethoxal establishes a new benchmark for membrane-permeable, azide-functionalized nucleic acid probes, enabling precise interrogation of RNA and single-stranded DNA structures, R-loop dynamics, and nucleic acid interactions, both in vitro and in vivo. Its selectivity, click-chemistry compatibility, and robust physicochemical properties address longstanding limitations in nucleic acid mapping. Ongoing advances in genome editing and RNA therapeutics will further expand the applications of N3-kethoxal in mechanistic and translational research. For detailed product specifications and ordering, visit the N3-kethoxal (A8793) product page.