Examination of cytidine deamination activity and sequence specificity of human and mouse APOBEC2, APOBEC3, and APOBEC4 enzymes using a synthetic single-stranded DNA/RNA with degenerate sequence segments and cytidines at fixed positions
To evaluate DNA mutation and RNA editing through cytidine (C) deamination by multiple human and mouse APOBEC2-4 enzymes and an APOBEC3G-like putative human pseudogene, in vitro deamination reactions were performed with a single-stranded DNA/RNA (Y439) with degenerate sequence segments and C residues at specific positions. Whole cell lysates of human 293T cells that had been transiently transfected with plasmid DNA to express one of the APOBECs were used as enzyme source in deamination reactions with a single-stranded (ss) DNA (Y439) or RNA (Y439) as substrate. After the reaction, the substrate was amplified by PCR/RTPCR and purified. Amplicon DNA concentration was quantified with Thermo Fisher Qubit and Agilent TapeStation assays. Sequencing libraries were prepared for the amplicon using Rubicon Genomics ThruPLEX DNA-Seq kit using 10 ng DNA and total 9 PCR cycles and quantified with KAPA Biosystems qPCR kit. Illumina NextSeq 500 with 75-cycle V2 Reagent kit was used for sequencing in a single run to obtain single-end 75-base reads (with dual 8-base index reads for demultiplexing); the multiplexed run had 1.8 pmole of each library and 20% PhiX. Transfection, cell lysate preparation, DNA or RNA deamination reaction, amplicon generation and purification, sequencing library preparation, and sequencing were all performed in a single batch. The raw sequencing data are merged lane-specific fastq.gz files. Sample/file-names are indicative of APOBEC, DNA/RNA substrate, deamination reaction duration, and combination of indexes used for sequencing library preparation. E.g., h2_D4ab = Human APOBEC2 transfectant lysate, DNA substrate, and 45-minute deamination reaction; with a and b indexes used for library indexing.  (A) Nucleic acid sequences:  - Y439: AGGGAAGGAGAGGTGAGATGATNNNNCNNNNTGAGGTTAGNNNNCNNNNTTGTGAGTANNNNCNNNNTAGGTATGGGTGGTAGTGTGAA - Y440: TTCAGGTAATACGACTCACTATAGGGAAGGAGAGGTGAGATG - Y441: AGGGAAGGAGAGGTGAGATG - Y442: TAGGGAAGGAGAGGTGAGATG - Y443: GTAGGGAAGGAGAGGTGAGATG - Y444: CCAAGGGAAGGAGAGGTGAGATG - Y445: ATCAAGGGAAGGAGAGGTGAGATG - Y446: TGCCTAGGGAAGGAGAGGTGAGATG - Y447: GATTCCAGGGAAGGAGAGGTGAGATG - Y448: CCTTCTCAGGGAAGGAGAGGTGAGATG - Y449: TTCACACTACCACCCATACC - Y450: GTTCACACTACCACCCATACC - Y451: CGTTCACACTACCACCCATACC - Y452: ACGTTCACACTACCACCCATACC - Y453: TACGTTCACACTACCACCCATACC - Y454: GTACGTTCACACTACCACCCATACC  All DNA were commercially synthesized by IDT. Y439 synthesis used "hand-mixed" bases.  (B) Transient transfectant cell lysate preparation: Adherent human HEK 293T cells were transfected with plasmid DNA using jetPRIME chemical transfection reagent (Polyplus). After ~2 days, whole cell lysates were prepared using M-PER detergent with 1x Halt protease & phosphatase inhibitor cocktail (200 ul M-PER + 2 ul Halt) – M-PER: Thermo Fisher product 78503; Halt: Thermo Fisher product 1861280. Protein concentration of lysate was measured. Lysates were frozen at -80 deg. C in aliquots and thawed for one-time, immediate use in deamination reactions.  (C) DNA deamination reaction: 24 ul total volume, with 11.1 ul of cell lysate made to 3.7 ug/ul protein using M-PER/Halt, and containing these ingredient stocks at final 1x in the reaction:  - 10x UDG reaction buffer (200 mM Tris-HCl, 10 mM EDTA, 10 mM DTT, pH 8) – New England Biolabs product B0280 - 80x RNAseA (2 U/ul; bovine pancreas) – Sigma - 10x ZnCl2 (1 mM) - 5x Y439 DNA (10 uM, in 0.1x Tris-EDTA [10 mM Tris, 1 mM EDTA, pH 8.0]) – heated at 98 deg. C for 4.5 min and immediately chilled on ice for 2 min. before addition to reaction - 8x UGI UDG inhibitor (2 U/ul) – New England Biolabs product M0281  Reactions were for 15 or 45 minutes at 37 deg. C and stopped by adding 4x volume water before being stored at -80 deg. C.  (D) Amplicon preparation from DNA deamination reaction: 9 ul of the -80-stored reaction was used as template in a 7-cycle PCR of 60 ul volume using Q5 hot-start DNA polymerase (New England Biolabs, product M0493) and 2 uM (final conc.) of PCR primers (equimolar mixes of Y441-448 and Y449-454 as forward and reverse primers respectively). Cycling was as 98 deg. C x 10 s, 65 deg. C x 20 s, and 72 deg. C x 10 s. PCR was electrophoresed on a 2.5% agarose/0.5x TB gel and gel piece with ~90-100 bp band for expected amplicon was cut (visualized under UV for < 40 s) for DNA isolation using Qiagen QIAquick Gel Extraction kit.  (E) RNA deamination reaction: 18 ul total volume, with 9 ul of cell lysate made to 3.7 ug/ul protein using M-PER/Halt, and containing these ingredient stocks at final 1x in the reaction:  - 100x HEPES (1 M, pH 7.5) – Corning product 25-060-CI - 10x ZnCl2 (1 mM) - 20x RNAse inhibitor (40 U/ul) – New England Biolabs product M0314 - 100x DTT (1 M) - 13.3x KCl (1 M) - 9x Y439 RNA (1 uM, in water) – heated at 70 deg. C for 6 min and immediately chilled on ice for 2 min. before addition to reaction  Reactions were for 15 or 45 minutes at 37 deg. C and stopped by adding 4x volume water before being stored at -80 deg. C.  Y439 RNA was made by first amplifying Y439 with Y440 (which has a T7 polymerase site) and Y449 with Q5 hot-start polymerase and purifying the product in similar fashion as described below. In vitro transcription reaction of 75 ul was then performed at 37 deg. C for 20 h on the purified DNA (1.5 ug) using T7 RNA polymerase (250 U, New England Biolabs product M0251) as per manufacturer-suggested protocol. The reaction was treated with DNAse I (60 U, Roche) for 2 h and RNA purified from it using Norgen Biotek Total RNA purification kit.  (F) Amplicon preparation from RNA deamination reaction: Norgen Biotek Total RNA Purification kit was used to extract RNA in 50 ul water from the deamination reactions. New England Biolabs's M-MuLV RT enzyme (product M0253) was used as per manufacturer protocol to prepare cDNA of 20 ul from 12 ul of RNA with Y449 primer. Amplicon preparation was like that described for DNA deamination reactions.
To evaluate DNA mutation and RNA editing through cytidine (C) deamination by multiple human and mouse APOBEC2-4 enzymes and an APOBEC3G-like putative human pseudogene, in vitro deamination reactions were performed with a single-stranded DNA/RNA (Y439) with degenerate sequence segments and C residues at specific positions. Whole cell lysates of human 293T cells that had been transiently transfected with plasmid DNA to express one of the APOBECs were used as enzyme source in deamination reactions with a single-stranded (ss) DNA (Y439) or RNA (Y439) as substrate. After the reaction, the substrate was amplified by PCR/RTPCR and purified. Amplicon DNA concentration was quantified with Thermo Fisher Qubit and Agilent TapeStation assays. Sequencing libraries were prepared for the amplicon using Rubicon Genomics ThruPLEX DNA-Seq kit using 10 ng DNA and total 9 PCR cycles and quantified with KAPA Biosystems qPCR kit. Illumina NextSeq 500 with 75-cycle V2 Reagent kit was used for sequencing in a single run to obtain single-end 75-base reads (with dual 8-base index reads for demultiplexing); the multiplexed run had 1.8 pmole of each library and 20% PhiX. Transfection, cell lysate preparation, DNA or RNA deamination reaction, amplicon generation and purification, sequencing library preparation, and sequencing were all performed in a single batch. The raw sequencing data are merged lane-specific fastq.gz files. Sample/file-names are indicative of APOBEC, DNA/RNA substrate, deamination reaction duration, and combination of indexes used for sequencing library preparation. E.g., h2_D4ab = Human APOBEC2 transfectant lysate, DNA substrate, and 45-minute deamination reaction; with a and b indexes used for library indexing.  (A) Nucleic acid sequences:  - Y439: AGGGAAGGAGAGGTGAGATGATNNNNCNNNNTGAGGTTAGNNNNCNNNNTTGTGAGTANNNNCNNNNTAGGTATGGGTGGTAGTGTGAA - Y440: TTCAGGTAATACGACTCACTATAGGGAAGGAGAGGTGAGATG - Y441: AGGGAAGGAGAGGTGAGATG - Y442: TAGGGAAGGAGAGGTGAGATG - Y443: GTAGGGAAGGAGAGGTGAGATG - Y444: CCAAGGGAAGGAGAGGTGAGATG - Y445: ATCAAGGGAAGGAGAGGTGAGATG - Y446: TGCCTAGGGAAGGAGAGGTGAGATG - Y447: GATTCCAGGGAAGGAGAGGTGAGATG - Y448: CCTTCTCAGGGAAGGAGAGGTGAGATG - Y449: TTCACACTACCACCCATACC - Y450: GTTCACACTACCACCCATACC - Y451: CGTTCACACTACCACCCATACC - Y452: ACGTTCACACTACCACCCATACC - Y453: TACGTTCACACTACCACCCATACC - Y454: GTACGTTCACACTACCACCCATACC  All DNA were commercially synthesized by IDT. Y439 synthesis used "hand-mixed" bases.  (B) Transient transfectant cell lysate preparation: Adherent human HEK 293T cells were transfected with plasmid DNA using jetPRIME chemical transfection reagent (Polyplus). After ~2 days, whole cell lysates were prepared using M-PER detergent with 1x Halt protease & phosphatase inhibitor cocktail (200 ul M-PER + 2 ul Halt) – M-PER: Thermo Fisher product 78503; Halt: Thermo Fisher product 1861280. Protein concentration of lysate was measured. Lysates were frozen at -80 deg. C in aliquots and thawed for one-time, immediate use in deamination reactions.  (C) DNA deamination reaction: 24 ul total volume, with 11.1 ul of cell lysate made to 3.7 ug/ul protein using M-PER/Halt, and containing these ingredient stocks at final 1x in the reaction:  - 10x UDG reaction buffer (200 mM Tris-HCl, 10 mM EDTA, 10 mM DTT, pH 8) – New England Biolabs product B0280 - 80x RNAseA (2 U/ul; bovine pancreas) – Sigma - 10x ZnCl2 (1 mM) - 5x Y439 DNA (10 uM, in 0.1x Tris-EDTA [10 mM Tris, 1 mM EDTA, pH 8.0]) – heated at 98 deg. C for 4.5 min and immediately chilled on ice for 2 min. before addition to reaction - 8x UGI UDG inhibitor (2 U/ul) – New England Biolabs product M0281  Reactions were for 15 or 45 minutes at 37 deg. C and stopped by adding 4x volume water before being stored at -80 deg. C.  (D) Amplicon preparation from DNA deamination reaction: 9 ul of the -80-stored reaction was used as template in a 7-cycle PCR of 60 ul volume using Q5 hot-start DNA polymerase (New England Biolabs, product M0493) and 2 uM (final conc.) of PCR primers (equimolar mixes of Y441-448 and Y449-454 as forward and reverse primers respectively). Cycling was as 98 deg. C x 10 s, 65 deg. C x 20 s, and 72 deg. C x 10 s. PCR was electrophoresed on a 2.5% agarose/0.5x TB gel and gel piece with ~90-100 bp band for expected amplicon was cut (visualized under UV for < 40 s) for DNA isolation using Qiagen QIAquick Gel Extraction kit.  (E) RNA deamination reaction: 18 ul total volume, with 9 ul of cell lysate made to 3.7 ug/ul protein using M-PER/Halt, and containing these ingredient stocks at final 1x in the reaction:  - 100x HEPES (1 M, pH 7.5) – Corning product 25-060-CI - 10x ZnCl2 (1 mM) - 20x RNAse inhibitor (40 U/ul) – New England Biolabs product M0314 - 100x DTT (1 M) - 13.3x KCl (1 M) - 9x Y439 RNA (1 uM, in water) – heated at 70 deg. C for 6 min and immediately chilled on ice for 2 min. before addition to reaction  Reactions were for 15 or 45 minutes at 37 deg. C and stopped by adding 4x volume water before being stored at -80 deg. C.  Y439 RNA was made by first amplifying Y439 with Y440 (which has a T7 polymerase site) and Y449 with Q5 hot-start polymerase and purifying the product in similar fashion as described below. In vitro transcription reaction of 75 ul was then performed at 37 deg. C for 20 h on the purified DNA (1.5 ug) using T7 RNA polymerase (250 U, New England Biolabs product M0251) as per manufacturer-suggested protocol. The reaction was treated with DNAse I (60 U, Roche) for 2 h and RNA purified from it using Norgen Biotek Total RNA purification kit.  (F) Amplicon preparation from RNA deamination reaction: Norgen Biotek Total RNA Purification kit was used to extract RNA in 50 ul water from the deamination reactions. New England Biolabs's M-MuLV RT enzyme (product M0253) was used as per manufacturer protocol to prepare cDNA of 20 ul from 12 ul of RNA with Y449 primer. Amplicon preparation was like that described for DNA deamination reactions.
ROSWELL PARK CANCER INSTITUTE