N3-kethoxal and its history

How was N3-kethoxal invented? AccuraDX's Scientific Co-Founder Professor Chuan He and his laboratory at The University of Chicago invented N3-kethoxal, also called azide kethoxal. The IUPAC name for azide kethoxal is 3-(2-azidoethoxy)-1,1-dihydroxybutan-2-one. The history of azide kethoxal is simple and elegant. It begins with di-aldehyde glyoxal, a metabolic product that is well known to covalently react with guanidine groups, such as those found in arginines and guanosines, by serving as an bi-functional electrophile. Kethoxal is a keto-aldehyde derivative of glyoxal, and has a higher selectivity for guanine and was established in the late 1950s as an antiviral agent. Leveraging the power of kethoxal to bind to single-stranded guanosines in RNA and DNA of living cells, the He laboratory team at The University of Chicago reasoned that the molecule could be used to perform genome-wide mapping of sites of single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA). But first, there needed to be a way to capture the nucleic acid fragments after they were bound by kethoxal. To do this, azide kethoxal was invented, a kethoxal derivative with an azide group to enable copper-free Click chemistry. The azide group is important to allow the kethoxal adducts of ssDNA or ssRNA to be biotinylated (for capture, amplification, and next-generation sequencing) by first reacting the azide moiety with a strained alkyne linked to biotin. Next, biotinylated fragments can then be affinity-captured by streptavidin beads. This rationale forms the basis for the innovative technologies keth-seq to reveal RNA secondary structure and KAS-seq (to reveal sites of ssDNA such as transcriptional bubbles and active enhancers. AccuraDX is excited to launch N3-kethoxal for researchers to use for their various ssDNA and ssRNA applications. AccuraDX has the exclusive commercialization rights to the azide kethoxal molecule and as a University of Chicago spin-out start-up company, we have collaborated closely with the inventors to assure that our formulation is the highest quality and developed specifically with KAS-seq and keth-seq in mind. Check out our azide kethoxal DMSO solution today!

Azide Kethoxal References:

Azide kethoxal in KAS-seq: Wu, T.; Lyu, R.; You, Q.; He, C. Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ. Nat. Methods 2020, 17, 515-523. https://doi.org/10.1038/s41592-020-0797-9 PMID: 32251394; PMCID: PMC7205578.

Azide kethoxal in Keth-seq: Weng, X.; Gong, J.; Chen, Y.; Wu, T.; Wang, F.; Yang, S.; Yuan, Y.; Luo, G.; Chen, K.; Hu, L.; Ma, H.; Wang, P.; Zhang, Q. C.; Zhou, X.; He, C. Keth-seq for transcriptome-wide RNA structure mapping. Nat. Chem. Biol. 2020, 16, 489-492. https://doi.org/10.1038/s41589-019-0459-3 PMID: 32015521; PMCID: PMC7182492.

Kethoxal and Glyoxal References: 

Staehelin, M. Inactivation of virus nucleic acid with glyoxal derivatives. Biochim Biophys Acta. 1959 Feb;31(2):448-54. doi: 10.1016/0006-3002(59)90019-8. PMID: 13628672.

Litt M, Hancock V. Kethoxal--a potentially useful reagent for the determination of nucleotide sequences in single-stranded regions of transfer ribonucleic acid. Biochemistry. 1967 Jun;6(6):1848-54. doi: 10.1021/bi00858a036. PMID: 6035923.

Noller HF, Chaires JB. Functional modification of 16S ribosomal RNA by kethoxal. Proc Natl Acad Sci U S A. 1972;69(11):3115-3118. doi:10.1073/pnas.69.11.3115