The Nucleoside Digestion Mix is an optimized mixture of enzymes that provides a convenient one-step method to generate single nucleosides from DNA or RNA for quantitative analysis by liquid chromatography-mass spectrometry (LC-MS), eliminating the need for sequential multi-step, time-consuming digestion protocols.
Convenient one-step protocol
Digests both DNA and RNA to single nucleosides
Low-glycerol formulation significantly reduces glycerol-induced ion suppression during MS analysis
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.
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The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
M0649S
-20
Nucleoside Digestion Mix
M0649SVIAL
-20
1 x 0.05 ml
50 reactions
Nucleoside Digestion Mix Reaction Buffer
B0649SVIAL
-20
1 x 0.5 ml
10 X
Properties & Usage
Reaction Conditions
1X Nucleoside Digestion Mix Reaction Buffer
Incubate at 37°C
1X Nucleoside Digestion Mix Reaction Buffer
50 mM sodium acetate
1 mM ZnCl2
(pH 5.4 @ 25°C)
Storage Buffer
20 mM Tris-HCl
1 mM MgCl2
2 mM CaCl2
2 mM ZnCl2
50 mM NaCl
0.6% Glycerol
pH 7.5 @ 25°C
Product Notes
Dilution of the Nucleoside Digestion mix may result in a decrease of enzymatic activity and incomplete digestion of substrate. Therefore, we recommend using 1 µL of the Nucleoside Digestion Mix for digestion of samples containing < 1 µg of DNA or RNA substrate.
Samples containing a large number of modifications (particularly modifications at the ribose 2´-position) may benefit from overnight incubation with the Nucleoside Digestion Mix in order to achieve complete digestion. No signal deterioration has been observed by incubating DNA or RNA samples with the Nucleoside Digestion Mix for up to 24 hours at 37°C. Alternatively, the ratio of Nucleoside Digestion Mix:nucleic acid may be increased from 1 μl/μg substrate to 5-10 μl/μg substrate to ensure complete digestion.
Although it is not necessary to stop the reaction prior to LC-MS, the mix can be inactivated by the addition of EDTA (5 mM, final concentration).
In order to reduce the ion suppression effects of glycerol, the Nucleoside Digestion Mix has been formulated to contain very little glycerol (<1%) and therefore the mix will freeze when stored at -20°C. The mix is stable for >2 years when stored at -20°C and can withstand 50 freeze-thaw cycles without significant activity loss.
As carryover of certain reaction components (e.g., EDTA, detergents, etc) from upstream steps may result in incomplete digestion, it is highly recommended that DNA or RNA substrates be purified (column purification/phenol chloroform extraction) before digestion with the Nucleoside Digestion Mix.
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
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
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
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
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