Dnase 1

Before determination of genomic titers via qPCR, samples were treated with 10 U DNase I (New England Biolabs, Frankfurt am Main, Germany) in 10× DNaseI buffer in a final volume of 50 µL at 37 °C for 30 min before heat inactivation of the DNase I (75 °C, 20 min). Crude lysate samples were additionally incubated with 0.8 U Proteinase K (New England Biolabs) for 50 min at 37 °C before heat inactivation (95 °C, 10 min). Dilutions of the DNase I digest were used as template in the qPCR reaction. The sample was mixed with 2.5 µL primer qPCR-hGH-for (5′-CTCCCCAGTG CCTCTCCT-3′) and 2.5 µL primer qPCR-hGH-rev (5′-ACTTGCCCCT TGCTCCATAC-3′), each at a stock concentration of 4 µM, and 10 µL of 2 × GoTaq qPCR Mastermix (Promega, Mannheim, Germany). The qPCR reaction was carried out as described in the manual (TM318 6/14, Promega) with an increased time interval for the first denaturation step (95 °C, 10 min) using a LightCycler 480 II (Roche, Mannheim, Germany). The genomic titer was calculated from a standard curve of 10² to 10⁷ copies of the ITR plasmid (pZMB0522) with an efficiency between 90% and 110% and an R value less than 0.1. Genomic titers in crude lysates were estimated from a standard curve mixed with the same amount of a non-transfected cell lysate.

APOBEC-YTH and APOBEC-YTH^mut were purified and in vitro DART-Sanger sequencing assays were performed as previously described (Tegowski et al., 2022 (link)) with minor modifications. Briefly, total RNA was isolated from adult heads with TRIzol (Invitrogen) and treated with DNase I (NEB). RNA was isolated once more with TRIzol (Invitrogen) to remove DNase I and DNase I Buffer (NEB). Next, 200 ng of purified RNA from Drosophila heads was incubated with 1000 ng of purified DART protein in DART buffer (10 mM Tris-HCl, pH 7.4, 50 mM KCl, 0.1 M ZnCl₂) and 1 µL of RNaseOUT (Invitrogen) in a total volume of 200 µL for 4 hr at 37°C. RNA was isolated with the QIAGEN Plus Micro Kit (QIAGEN) and stored at –80°C before being thawed for downstream Sanger sequencing analysis. cDNA was made using iScript Reverse Transcription Supermix (Bio-Rad). PCR amplification of Sxl pre-mRNA was carried out with Phusion High Fidelity PCR Kit (NEB). The resulting PCR product was PCR-purified using the QIAGEN PCR Purification Kit (QIAGEN). Samples were submitted for Sanger sequencing (McLabs) and %C-to-U editing was quantified using EditR software (Kluesner et al., 2018 (link)).

Agarose gel electrophoresis was used to study DNA binding and DNase protection afforded by lipoplex-A to plasmid DNA. Increasing weight ratios of lipoplex-A (1:1, 1:5, 1:10, 1:50, and 1:100 of DNA:SLN-A w/w) were incubated overnight at 4 °C. After binding, 10 µL of each sample was run per lane on a 1% agarose gel at 100 kV for 60 min. To determine the ability of the SLN-A to protect bound plasmid DNA from nuclease degradation, a DNase protection assay was performed using the same samples, which were treated with 1 U/µg of DNase I (New England Biolabs, Ipswich, MA, USA) for 30 min at 37 °C followed by heat inactivation of DNase I at 75 °C for 10 min. Following this, the remaining DNA was released from the lipoplexes by the addition of 10 µL of 1% SDS and samples were run on a 1% agarose gel to visualise the quantity of undigested plasmid DNA. Treated pcDNA3.1-UreA served as an internal positive control for DNase digestion.

Total RNA was isolated by extraction with hot acidic-phenol (pH-5) as described by Collart and Oliviero [94 ]. For RNA half-life experiments, cells were grown in SC medium- to mid-log phase at permissive temperature (25°C), centrifuged and shifted rapidly to non-permissive temperature (39°C) by adding equal amount of pre-warmed medium (50°C) and incubated at 39°C for the respective intervals. For rat1–1 ts mutant, the cells were sub-cultured and grown up to OD₆₀₀ 0.5 ~ 0.8 (28°C) and shifted to non-permissive temperature (39°C) for 3 hours. For data normalization of rat1–1, similarly treated wild-type cells were used. For DNaseI digestion, 3 μg of RNA was subjected to digestion with 10 units of DNaseI (New England Biolabs) enzyme at 37°C for 3.5 hours to achieve an RNA which is completely devoid of telomeric DNA. Before c-DNA synthesis, a normal PCR (40 Cycles) was performed with primers specific for telomere and ACT1 to assess the DNA contamination. RNA alone was also employed as a template for qRT-PCR to confirm the purity of the samples. The reverse transcription was performed using Verso cDNA Synthesis Kit (Thermo Scientific) as per the manufacturer’s protocol at 55°C for 60 min followed by inactivation at 95°C for 2 min.

Cy5-labeled 14 mM MgCl₂ DNA nanotube cytoskeleton DMPC/cholesterol protocells (60 µL) were added to 1 Chol strand containing a FAM fluorophore (5 µL, 25 µM, in water) and left to incubate at 37 °C for 15 min. Next, either hairy DNA nanotubes or hairy fibers (1 µL, 100 µM, in 14 mM MgCl₂, 1× TAE pH 8.3) were added and the solution left for 15 min. The exoskeleton GUVs (2 µL) were then deposited into PBS (10 µL) and imaged using CLSM. For the nuclease digestion assay, DNase I (1 µL, 1 mg per mL) (New England Biolabs, UK) in 1× DNase I buffer was carefully added to the top of the droplet and a time series performed, scanning every 10 s. Digestion profiles for cyto- and exoskeletons were identified by plotting the fluorescence intensities over time, profiles were obtained from a single time series.

When noted, phage titers were estimated by qPCR rather than plaque assay. SP pools (50 µl) were first heated at 80 °C for 30 min to destroy polyphage. Polyphage genomes were then degraded by adding 5 µl of heated SP to 45 µl of 1× DNase I buffer containing 1 µl of DNase I (New England Biolabs) and incubated at 37 °C for 20 min followed by 95 °C for 20 min. Next, 1.5 µl of each prepared phage DNA stock was then added to a 25 µl of qPCR reaction, prepared as follows: 10.5 µl of H₂O, 12.5 µl 2× Q5 Mastermix (New England Biolabs), 0.25 µl of Sybr Green (Thermo Fisher Scientific), 0.125 µl of each primer (qPCR forward, 5′-CACCGTTCATCTGTCCTCTTT and qPCR reverse, 5’-CGACCTGCTCCATGTTACTTAG, Supplementary Table 7). qPCR was then run with the following cycling conditions: 98 °C for 2 min, 45 cycles of 98 °C for 10 s, 60 °C for 20 s and 72 °C for 15 s. Titers were calculated using a titration curve of an SP standard of known titer (by plaque assay). A limit of detection was set based on when primers amplified (without SP) or at the lowest titer before loss of linearity for the SP standard.