DNA editing in DNA/RNA hybrids by adenosine deaminases that act on RNA

Abstract

Adenosine deaminases that act on RNA (ADARs) carry out adenosine (A) to inosine (I) editing reactions with a known requirement for duplex RNA. Here, we show that ADARs also react with DNA/RNA hybrid duplexes. Hybrid substrates are deaminated efficiently by ADAR deaminase domains at dA-C mismatches and with E to Q mutations in the base flipping loop of the enzyme. For a long, perfectly matched hybrid, deamination is more efficient with full length ADAR2 than its isolated deaminase domain. Guide RNA strands for directed DNA editing by ADAR were used to target six different 2΄-deoxyadenosines in the M13 bacteriophage ssDNA genome. DNA editing efficiencies varied depending on the sequence context of the editing site consistent with known sequence preferences for ADARs. These observations suggest the reaction within DNA/RNA hybrids may be a natural function of human ADARs. In addition, this work sets the stage for development of a new class of genome editing tools based on directed deamination of 2΄-deoxyadenosines in DNA/RNA hybrids.

Figure 1.

Interactions between hADAR2d and 2΄-hydroxyl groups. (A) Structure of hADAR2d bound to duplex RNA (14). The edited strand is colored salmon with the unedited strand colored slate. The editing site nucleotide is shown in red in its flipped out conformation. (B) 2΄-Hydroxyl contacts to the RNA substrate in the crystal structure (PDB: 5ED2).

Figure 2.

Deamination kinetics for an RNA duplex and partially 2΄-deoxyribose-substituted substrates. (A) Sequences of deamination substrates. 2΄-Deoxynucleotides are labeled in red. Target sites are underlined and bolded. (B) Comparison of deamination product versus time for the three substrates with 300 nM hADAR2d. (C) Kinetic parameters for the deamination of RNA and partially 2΄-deoxy substrates.

Figure 3.

Comparison of deamination reactions with DNA/RNA hybrid, all RNA and all DNA substrates. (A) Sequences of deamination substrates. Strands colored red are DNA and strands colored black are RNA. Editing sites are underlined and bolded (DD: both strands DNA, DR: edited strand DNA, complementary strand RNA; RD: edited strand RNA, complementary strand DNA; RR: both strands RNA. Strands containing the edited A are underlined.). (B–E) Percent editing for deamination reactions at different time points with 250 nM of hADAR1d, hADAR1d E1008Q, hADAR2d and hADAR2d E488Q. Statistical significance between groups was determined by t tests. (***P-value ≤ 0.001, **P-value ≤ 0.01, *P-value ≤ 0.05). (F) Deamination reaction yield vs. time for 250 nM hADAR2 wt. Kinetic parameters (k_obs): DD: k_obs ≤ 0.001 min⁻¹, DR: k_obs = 0.02 min⁻¹, RD: k_obs = 0.02 min⁻¹, RR: k_obs ≥ 0.4 min⁻¹ (n = 2, reported value is the average from two trials).

Figure 4.

Deamination in the DNA strand of DNA/RNA hybrid duplexes; effects of mismatches and duplex length. (A) Sequence surrounding three editing sites (A, B and C). Red color indicates DNA. Black corresponds to sequence of 24 nt RNA bottom strand with varying X and Y positions. Gray corresponds to 93 nt RNA bottom strand. (B–F) Percent editing for sites A, B and C in the different substrate structures shown with either hADAR2 wt or hADAR2d E488Q. Statistical significance between groups was determined by t tests. (***P-value ≤ 0.001, **P-value ≤ 0.01, *P-value ≤ 0.05).

Figure 5.

Selective editing of multiple sites in the ssDNA genome from M13 bacteriophage. (A) Single-stranded DNA and six target sites. Target sites are shown in red. Guide RNAs are shown in green. (B) Percent editing by 500 nM hADAR1d E1008Q at each site. (C) Off target site found adjacent to AAT target. Off target site is marked with an asterisk in sequence trace. Statistical significance between groups was determined by t tests (***P-value ≤ 0.001, **P-value ≤ 0.01, *P-value ≤ 0.05).