Journal: bioRxiv

Article Title: Synthetic circRNAs employ IRES activity for translation in cells and in cell-free translation systems

doi: 10.64898/2026.03.28.715045

Figure Lengend Snippet: (A) Experimental outline of the in vitro generated circRNA-based reporter assay based on the mRuby3_circ_EGFP_ScaI_v3-4 plasmid (pKL480) for evaluation of IRES activity of different IRES inserts, including inverse sequences as controls for circRNA translation dependent on insert length and GC-content. Following DNA template synthesis (PCR amplification from plasmid backbone using poly(A)- and poly(T)-including primers), circRNA was generated by T7 in vitro transcription. Linear RNA species were removed by RNase R-treatment and column purification. Resulting circRNA was subsequently used for in-cell transfection or for in vitro translation systems. EGFP and 3xHA-Nluc reporter systems were used throughout the following analyses. The plasmid-encoded mRuby was not used. (B) Illustration of the circularization reaction mediated by group I dt introns leading to self-spliced circRNA. The observed sequence scar formed by the remaining parts of the td introns are highlighted in light and dark red. Remaining split introns were generated as side products and need to be removed. (C) Overview of the tested IRES sequences indicating length and viral or cellular origin of the IRESes. (D) Quality control of the generated EGFP reporter including circRNAs using 1% FA-agarose gel. Linear side products (upper band) disappear by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). 500 ng total RNA was loaded per lane. The RiboRuler High Range RNA Ladder (Thermo, #SM1821) serves as a reference. (E) Quality control of the generated 3xHA-Nluc encoding circRNAs using 1% FA-agarose gel. Linear side products (upper band) are mainly degraded by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). Only Dlx1 shows less efficient circularization leading to a higher concentration of side products. Therefore, the RNase R incubation time was increased to 90 min. 500 ng total RNA was loaded per lane. RNA species: linear (L), circular (C), introns (I). (F) Quality control, using the High Sensitivity RNA ScreenTape, of the generated circRNA before purification (upper panel) and after RNase R digestion (lower panel). Remaining contaminants can be observed as light grey bands.

Article Snippet: The PCR products were purified using the Monarch PCR & DNA Cleanup kit (NEB, T1030L) and 1 μg DNA was subsequently used for in vitro transcription using the High Scribe T7 High Yield RNA synthesis Kit (NEB, E2040S) according to manufacturer’s protocol (20 μL total reaction volume) supplemented with 40 U RiboLock RNase inhibitor (Thermo, EO0381) and incubated for 2 hrs at 37°C and 800 rpm.

Techniques: In Vitro, Generated, Reporter Assay, Plasmid Preparation, Activity Assay, Amplification, Purification, Transfection, Sequencing, Control, Agarose Gel Electrophoresis, Concentration Assay, Incubation

Journal: bioRxiv

Article Title: ChromSMF: integrated profiling of histone modifications, protein-DNA interactions and DNA methylation on multi-kilobase DNA molecules

doi: 10.64898/2026.03.11.710921

Figure Lengend Snippet: (A) Schematic representation of the two-step ChromSMF protocol . Cells are chemically permeabilized and incubated with M.CviPI (cytosine-MTase), a methyltransferase that deposits cytosine methylation at accessible GpCs across the genome (black spheres). The obligatory cofactor SAM is washed out to stop M.CviPI activity. Cells are then sequentially incubated with an antibody (Ab) targeting a histone modification, and the proteinA-Hia5 (adenine-MTase) fusion protein that will methylate adenines in proximity of the histone modification of interest (green spheres). Sequencing of the resulting DNA using Oxford Nanopore Technologies (ONT) enables simultaneous detection of histone modifications (green signal; mA) and transcription factor binding events (black signal; mC) at bulk and single-molecule resolution. (B) Example locus illustrating the simultaneous detection of H3K4me3 and chromatin accessibility at active promoters . Top panel: genome browser tracks displaying ChIP-seq enrichment for H3K4me3 (green) and DNase-seq signal (black). Lower panel: ChromSMF sample for H3K4me3. Average SMF signal (1 – mC%) of individual cytosines (black) and average smoothed mA signal (mA%; green; smoothing across 4 adenines). (C) Simultaneous detection of chromatin accessibility and H3K4me3 on individual DNA molecules at a locus with low ChIP-seq enrichment for H3K4me3 . Single-molecule stacks display either mA-H3K4me3 (green, left) or mC-chromatin accessibility (black, right) signal. Molecules are displayed in identical order in both panels and originate from the same sample. Single-molecule classification of H3K4me3 (green) and chromatin accessibility (black) are shown as stacked bar plots between the single-molecule stacks. Single-molecule quantification of total H3K4me3 and chromatin accessibility at the locus are shown at the bottom. (D) Simultaneous detection of chromatin accessibility and H3K4me3 on individual DNA molecules at a locus with high ChIP-seq enrichment for H3K4me3 . Single-molecule stacks display either mA-H3K4me3 (green, left) or mC-chromatin accessibility (black, right) signal. Molecules are displayed in identical order in both panels and originate from the same sample. Single-molecule classification of H3K4me3 (green) and chromatin accessibility (black) are shown as stacked bar plots between the single-molecule stacks. Single-molecule quantification of total H3K4me3 and chromatin accessibility at the locus are shown at the bottom.

Article Snippet: Fully methylated CpG and GpC gDNA (sample 4) was generated by two consecutive 30 min incubations at 37°C with 8 U/μg DNA of M.CviPI GpC methyltransferase (in-house production) supplemented with 1.2 mM SAM (NEB, B9003S), followed by one 30 min incubation at 37°C with 16 U/μg DNA of M.SssI CpG methyltransferase (NEB, M0226L) in the presence of 7.5 μM MgCl2 and 0.8 mM SAM (NEB, B9003S).

Techniques: Incubation, Methylation, Activity Assay, Modification, Sequencing, Binding Assay, ChIP-sequencing