The Nucleoside Digestion Mix is a mixture of enzymes that provides a convenient one-step method to generate single nucleosides from DNA or RNA. Optimized for quantitative analysis by liquid chromatography-mass spectrometry (LC-MS), this reagent eliminates the need for sequential multi-step, time-consuming digestion protocols. The Nucleoside Digestion Mix digests ssDNA, dsDNA, DNA/RNA hybrids and RNA (except mRNA cap structures) containing epigenetically modified (m5C, hm5C, f5C, ca5C, m4C, m6A, etc.), unnatural, or damaged bases. Moreover, the low-glycerol formulation (<1%) significantly reduces glycerol-induced ion suppression during mass spectrometry analysis.
Supplied in: 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 2 mM CaCl2, 2 mM ZnCl2, 50 mM NaCl and 0.6% glycerol.
Figure 1. Global nucleoside analysis of HeLa DNA following incubation with the Nucleoside Digestion Mix Representative HPLC chromatogram of individual deoxyribonucleosides obtained from incubation of 1 µg of purified genomic HeLa DNA digested with 1 µl of the Nucleoside Digestion Mix for 1 hour at 37°C. Deoxyribonucleosides were separated by reversed-phase HPLC and detected by UV absorbance at 260 nm.
Figure 2. Global nucleoside analysis of HEK 293 RNA following incubation with the Nucleoside Digestion Mix
Representative HPLC chromatogram of individual ribonucleosides obtained from incubation of 1 µg of purified genomic HEK 293 RNA digested with 1 µl of the Nucleoside Digestion Mix overnight at 37°C. Ribonucleosides were separated by reversed-phase HPLC and detected by UV absorbance at 260 nm. The insert shows an expansion of the chromatogram highlighting the detection of modified ribonucleosides.
Figure 3. The activity of the Nucleoside Digestion Mix remains stable even after 50 freeze-thaws
After undergoing multiple freeze-thaw cycles, 1 µl of the Nucleoside Digestion Mix was used to digest 1 µg of λ DNA (NEB #N3011) for 1 hour at 37°C. The nucleoside content of the digested samples was analyzed by LC-MS.
This product is related to the following categories:
Yang, W., Lin, Y-C., Johnson, W., Dai, N., Vaisvila, R., Weigele, P., Lee, Y.-J., Corrêa, I.R., Jr., Schildkraut, I., Ettwiller, L. (2021) A genome-phenome association study in native microbiomes identifies a mechanism for cytosine modification in DNA and RNA. eLife; 10: e70021, PubMedID: 34747693, DOI: 10.7554/eLife.70021
Qi, S., Mota, J., Chan, S.-H., Villarreal, J., Dai, N., Arya, S., Hromas, R.A., Rao, M.K., Corrêa, I.R., Jr., Gupta, Y.K. (2022) RNA binding to human METTL3-METTL14 restricts N 6-deoxyadenosine methylation of DNA in vitro. Elife; 11, e67150. PubMedID: 35060905, DOI: 10.7554/eLife.67150
Vaisvila R, et al. (2021) Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA. Genome Res; PubMedID: 34140313, DOI: 10.1101/gr.266551.120
Abakir, A., Giles, T.C., Cristini, A., Foster, J.M., Dai, N., Starczak, M., Rubio-Roldan, A., Li, M., Eleftheriou, M., Crutchley, J., Flatt, L., Young, L., Gaffney, D.J., Denning, C., Dalhus, B., Emes, R.D., Gackowski, D., Corrêa, I.R., Jr., Garcia-Perez, J.L., Klungland, A., Gromak, N., Ruzov, A (2019) N6 methyladenosine regulates the stability of RNA DNA hybrids in human cells Nat Genet; PubMedID: 31844323, DOI: 10.1038/s41588-019-0549-x
Yang, W., Lin, Y-C., Johnson, W., Dai, N., Vaisvila, R., Weigele, P., Lee, Y.-J., Corrêa, I.R., Jr., Schildkraut, I., Ettwiller, L. (2021) A genome-phenome association study in native microbiomes identifies a mechanism for cytosine modification in DNA and RNA. eLife; 10: e70021, PubMedID: 34747693, DOI: 10.7554/eLife.70021
Vaisvila R, et al. (2021) Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA. Genome Res; PubMedID: 34140313, DOI: 10.1101/gr.266551.120
Qi, S., Mota, J., Chan, S.-H., Villarreal, J., Dai, N., Arya, S., Hromas, R.A., Rao, M.K., Corrêa, I.R., Jr., Gupta, Y.K. (2022) RNA binding to human METTL3-METTL14 restricts N 6-deoxyadenosine methylation of DNA in vitro. Elife; 11, e67150. PubMedID: 35060905, DOI: 10.7554/eLife.67150
Abakir, A., Giles, T.C., Cristini, A., Foster, J.M., Dai, N., Starczak, M., Rubio-Roldan, A., Li, M., Eleftheriou, M., Crutchley, J., Flatt, L., Young, L., Gaffney, D.J., Denning, C., Dalhus, B., Emes, R.D., Gackowski, D., Corrêa, I.R., Jr., Garcia-Perez, J.L., Klungland, A., Gromak, N., Ruzov, A (2019) N6 methyladenosine regulates the stability of RNA DNA hybrids in human cells Nat Genet; PubMedID: 31844323, DOI: 10.1038/s41588-019-0549-x
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