New England Biolabs, M0648S, APOBEC3A

Related Categories Base Modifying Enzymes,, DNA Methylation Analysis,, DNA Modifying Enzymes & Cloning Technologies Specification Materials Required but not Supplied dC- or 5mC-containing ssDNA Formamide (for dsDNA denaturation if needed) Microcentrifuge tubes Thermocycler or heat block Unit Definition One unit is defined as the amount of enzyme required to deaminate 5 pmol of a fluorescently labeled 44-mer oligonucleotide containing a single cytosine base in 30 minutes at 37°C in a total reaction volume of 20 μl in 1X Deamination Reaction buffer. Storage Temperature -20°C Reaction Conditions 1X Deamination Reaction Buffer Incubate at 37°C Heat Inactivation 70°C for 10 minutes Unit Assay Conditions 5 pmol of fluorescently labeled 44-mer oligonucleotide containing a single cytosine is incubated with 1 unit of APOBEC3A for 30 minutes at 37°C in a total reaction volume of 20 μl in 1X Deamination Reaction Buffer. The enzyme generated uracil-containing ssDNA is then incubated with USER enzyme in 1X rCutSmart Buffer to cleave the phosphodiester backbone at the uracil site. The resulting product is measured by capillary electrophoresis to determine APOBEC3A deamination activity. FAQ Q: What is the activity of APOBEC3A (NEB #M0648) in different NEBuffers? A: Relative to its optimal activity in Deamination Reaction Buffer, APOBEC3A has activity in the following reaction buffers: NEBuffer r1.1 25% NEBuffer r2.1 <10% NEBuffer r3.1 0% rCutSmart™ Buffer <10% Q: Can APOBEC3A (NEB #M0648) be heat inactivated? A: Yes, APOBEC3A can be inactivated at 70°C for 10 minutes. APOBEC3A can also be removed by treatment with Thermolabile Proteinase K (NEB #P8111). Q: What is the molecular weight of APOBEC3A (NEB #M0648)? A: The molecular weight of APOEBEC3A is 23,856 Daltons. Q: Is APOBEC3A (NEB #M0648) a tagged protein? A: APOBEC3A has a C-terminal His tag. Q: What is the relative activity of APOBEC3A (NEB #M0648) on different modified dC-containing ssDNA substrates? A: While APOBEC3A has a preferred substrate of C-containing ssDNA, it will deaminate 5mC-containing ssDNA with 2-fold less activity in a 30-minute reaction under recommended reaction conditions. In an overnight incubation (16 hours), APOBEC3A can reach complete deamination of 5mC-containing ssDNA and can deaminate 5-hydroxymethylcytosine (5hmC)-containing ssDNA less than 10%. APOBEC3A is not active on ssDNA containing beta-glucosyl-5-hydroxymethylcytosine (5gmC), 5-carboxylcytosine (5caC), or 5-formylcytosine (5fC). ssDNA Modification Activity after 30 min. Activity after 16 h C to U 100% 100% 5mC to T 50% 100% 5hmC to 5hmU 0% <10% 5fC to 5fU 0% 0% 5caC to 5caU 0% 0% 5gmC to 5gmU 0% 0% Activity on modified C ssDNA substrates was determined by incubating 1 µl of APOBEC3A with 1 μg (73 pmol) of a 44-mer DNA oligo containing a single C or modified C at 37°C in a total reaction volume of 40 µl in 1X Deamination Reaction Buffer. Samples were heat inactivated at each time point then analyzed by LCMS. Q: Which DNA modifications protect cytosine from deamination by APOBEC3A (NEB #M0648)? A: 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), and beta-glucosyl-5-hydroxymethylcytosine (5gmC) are resistant to deamination by APOBEC3A. Q: Is APOBEC3A (NEB #M0648) active on double stranded DNA? A: APOBEC3A is not recommended for dsDNA substrates. Activity on dsDNA was determined to be less than 10% by incubating 1 µl of APOBEC3A with 5 pmol of C-containing 44-mer dsDNA for 30 min. at 37°C in a total reaction volume of 20 µl in 1X Deamination Reaction Buffer. For dsDNA substrates, we recommend denaturation of dsDNA to ssDNA before deamination by adding formamide to the dsDNA (in water) and incubating at 85°C for 10 minutes, followed by immediate cooling on ice before proceeding to the APOBEC3A reaction. Alternatively, dsDNA can be denatured by adding 4 µl of 0.05 N NaOH to dsDNA (in water) and incubating at 85°C for 10 minutes, followed by immediate cooling on ice before proceeding to the APOBEC3A reaction. Q: Is APOBEC3A (NEB #M0648) active on RNA? A: While APOBEC3A has previously been shown to be active on RNA1,2, we do not observe any APOBEC3A activity on an RNA substrate under our tested conditions (1 μg (156 pmol) of C-containing 20-mer RNA is incubated with 16 units of APOBEC3A for 2 hours at 37°C in 1X Deamination Reaction Buffer). Therefore, we do not recommend APOBEC3A for deamination of RNA substrates. Barka, A. et. al. The Base-Editing Enzyme APOBEC3A Catalyzies Cytosine Deamination in RNA with Low Proficiency and High Selectivity. (2022) ACS Chem Biol. 17 (3) 629-636. https://doi.org/10.1021/acschembio.1c00919. Sharma, S. et al. APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages. (2015) Nat Commun. 6, 6881. https://doi.org/10.1038/ncomms7881. Q: Does APOBEC3A (NEB #M0648) exhibit any sequence preference? A: Yes, APOBEC3A has sequence preference for TC > CC > GC > AC 1, 2, 3, where the underlined C is the targeted cytosine for deamination in the dinucleotide. In order to provide robust activity on all substrates, the least preferred substrate, an AC motif, was used for the unit definition. Silvas, TV. et. al. Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions. (2018) Sci Rep. 8, 7511. https://doi.org/10.1038/s41598-018-25881-z. Stenglein, M. et. al. APOBEC3 proteins mediate the clearance of foreign DNA from human cells. (2010) Nat. Struct. Mol. Biol. 14, 222-229. https://doi.org/10.1038/nsmb.1744. Vartanian, JP. et. al. Evidence for Editing of Human Papillomavirus DNA by APOBEC3 in Benign and Precancerous Lesions. (2008) Science. 320, (5873) 230-233. https://doi.org/10.1126/science.1153201. Q: What temperatures is APOBEC3A (NEB #M0648) active? A: APOBEC3A is active across a temperature range of 25-50°C, but it is recommended for deamination activity at 37°C.