e coli poly a polymerase  (TaKaRa)

Journal: Nucleic Acids Research

Article Title: Generation of precise and accurate engineered circRNAs using enzymatic ligation

doi: 10.1093/nar/gkag405

Figure Lengend Snippet: Evaluation of circularization efficiency and accuracy by different ligases. ( A ) Schematic depicting requirements and features of DNA ligase, RNA ligase 1, and RNA ligase 2. ( B ) Workflow of circRNA generation using enzymatic ligation and RNase R based purification which can be improved by addition of poly(A) tails to linear RNAs. ( C ) 3% urea–PAGE showed that all ligases were able to circularize 5′-monophosphate RNAs. Boxed bands depict the circRNAs that run slower than their linear counterparts. Contaminating RNAs of lower and higher size than circular or linear RNA were also observed suggesting that poly(A) tailing and RNase R treatments were insufficient to degrade them. Efficiency of ligation was calculated as percentage yields of RNAs remaining after all treatments divided by input RNA for each ligation reaction. CircRNAs derived from modified transcription templates (mod) had higher efficiencies particularly for DNA ligase and RNA ligase 2 than those derived from unmodified templates (unmod). RNA ligase 2 had the highest circularization efficiency, especially with circRNAs derived from mod templates in presence of an RNA splint. Representative data are from a mean of n = 3 technical replicates with SEM; (*) P ≤.05 (unpaired t -test). ( D ) Sanger sequencing of ligation junctions showed accurate sequences with circRNAs derived from modified transcription templates using all ligases. CircRNAs made with RNA Ligase 1 had errors with the linear RNAs from unmodified templates which were corrected with the use of modified templates.

Article Snippet: Five micrograms of RNA were poly(A)-tailed using 5 units of Escherichia coli poly(A) polymerase (NEB #M0276L) for 30 min at 37°C.

Techniques: Ligation, Purification, Derivative Assay, Modification, Sequencing

Journal: Nucleic Acids Research

Article Title: Generation of precise and accurate engineered circRNAs using enzymatic ligation

doi: 10.1093/nar/gkag405

Figure Lengend Snippet: Purification of circRNAs and extending the RNA ligase 2 (RL2)-dependent circularization method to other RNAs. ( A ) CircRNAs synthesized with RNA ligase 2 using DNA splint were purified using three different approaches: from 3% urea–PAGE using crush and soak method, or from EX E-gels either using the crush and soak method or using column-based kit. As a control, linear RNAs were also extracted using the same methods. CircRNAs extracted from 3% urea–PAGE or EX E-gel using a crush and soak method had more intact circRNAs with less nicking compared to those extracted from EX E-gel using column-based kits. Linear RNAs on the other hand remained intact with each of the approaches. ( B ) Schematic of RNase-H based circularity confirmation assay that uses a short ssDNA probe which cleaves intact circRNAs into a single linear band, while nicked circRNAs or linear RNAs are cut into two shorter bands. ( C ) RNase-H based assay confirmed circularity of EGFP-IRES circRNAs. Linear RNAs were cleaved into two shorter bands of expected sizes while circRNAs derived from modified DNA templates were linearized to the size of full-length linear precursor. ( D, E ) 5′-monophosphate linear precursors of human immunodeficiency virus (HIV) and mCherry were ligated using RNA ligase 2 and respective DNA splints. For circHIV, urea–PAGE purification of circRNAs derived from modified templates had the highest yields with the least contaminating RNAs. Yields of mCherry circRNAs were much higher with polyA + RNase R approach on RNAs from modified template ligated using RNA ligase 2, however urea–PAGE showed higher and lower sized undesired RNAs. Representative data are from a mean of n = 3 technical replicates with SEM. ( F ) Sanger sequencing confirmed accuracy of circRNAs. Clean chromatograms were observed for ligation junctions of both HlV and mCherry circRNAs derived from modified DNA templates purified either through poly(A) tailing and RNase R treatment or from urea–PAGE purification.

Article Snippet: Five micrograms of RNA were poly(A)-tailed using 5 units of Escherichia coli poly(A) polymerase (NEB #M0276L) for 30 min at 37°C.

Techniques: Purification, Synthesized, Control, Rnase H Assay, Derivative Assay, Modification, Virus, Sequencing, Ligation

Journal: RSC Chemical Biology

Article Title: Enhancing mRNA stability and translational potential through tailored modifications at the 3′ end

doi: 10.1039/d6cb00033a

Figure Lengend Snippet: Modification and evaluation of Gaussia Luciferase mRNA. (A) 3′ End of GLuc mRNA is modified with different dinucleotides via ligation. (B) PAGE analysis of modified mRNAs, along with unmodified control GLuc mRNA, after 30 minutes of incubation with CNOT7. All modified mRNAs remained stable and full-length after incubation with CNOT7, whereas unmodified GLuc was shortened due to the removal of its poly(A) tail. Samples were denatured for 5 minutes at 75 °C prior to analysis on 7% polyacrylamide gel with urea in TBE buffer. The separation gel was stained with SYBR Gold before scanning. (C) Table of abbreviations.

Article Snippet: The poly(A) tail of mRNA was elongated to approximately 150 adenosines using the poly(A) Polymerase Tailing Kit (LuciGen) and following the standard protocol suggested by the manufacturer.

Techniques: Modification, Luciferase, Ligation, Control, Incubation, Staining

Journal: bioRxiv

Article Title: Benchmarking Tools for Identification of rRNA Modifications in Escherichia coli using Oxford Nanopore Direct RNA Sequencing

doi: 10.64898/2026.04.15.718756

Figure Lengend Snippet:

Article Snippet: We polyadenylated total RNA to enable direct RNA sequencing library preparation using the E. coli poly(A) polymerase kit (New England Biolabs, cat# M0276).

Techniques: Modification

Journal: bioRxiv

Article Title: Benchmarking Tools for Identification of rRNA Modifications in Escherichia coli using Oxford Nanopore Direct RNA Sequencing

doi: 10.64898/2026.04.15.718756

Figure Lengend Snippet: Heatmaps show the percentage of evaluated positions for which each tool reports a score, across 25 coverage depths (5x-1000×) on E. coli 16S rRNA (top) and 23S rRNA (bottom). Colour scale ranges from red (0%) through yellow (50%) to green (100%). Grey cells with em-dash indicate coverages where a tool failed to produce output and in the case of the tool nanoDoc, it indicates coverages not tested

Article Snippet: We polyadenylated total RNA to enable direct RNA sequencing library preparation using the E. coli poly(A) polymerase kit (New England Biolabs, cat# M0276).

Techniques:

Journal: bioRxiv

Article Title: Benchmarking Tools for Identification of rRNA Modifications in Escherichia coli using Oxford Nanopore Direct RNA Sequencing

doi: 10.64898/2026.04.15.718756

Figure Lengend Snippet: Performance of ten tools evaluated at 21 directional offsets (δ = -10 to +10 nt) from known modification sites on E. coli 16S and 23S rRNA at 1000× coverage. Negative δ values indicate upstream (5ʹ) displacement; positive δ values indicate downstream (3ʹ) displacement. (a) AUPRC across directional offset δ for each tool. Shaded bands indicate ±1 SD across replicates. The beige region highlights the ±2 nt range corresponding to the approximate 5-mer reader head context. (b) Row-normalised AUPRC heatmaps (each tool’s value scaled to its own maximum). White stars mark the peak offset per tool; the white vertical line indicates δ = 0 (true modification position). (c) Left: asymmetry index, defined as the difference between mean AUPRC over δ = +1 to +5 and mean AUPRC over δ = -1 to -5. Negative values indicate a 5ʹ bias (modification signal detected upstream of the true site). Right: peak offset δ that maximises AUPRC for each tool, shown separately for 16S (blue) and 23S (red) rRNA. Tools that peak at δ = 0 achieve exact positional localisation; tools peaking at negative δ values systematically mislocalise modification signals towards the 5ʹ direction.

Article Snippet: We polyadenylated total RNA to enable direct RNA sequencing library preparation using the E. coli poly(A) polymerase kit (New England Biolabs, cat# M0276).

Techniques: Modification

Journal: bioRxiv

Article Title: Benchmarking Tools for Identification of rRNA Modifications in Escherichia coli using Oxford Nanopore Direct RNA Sequencing

doi: 10.64898/2026.04.15.718756

Figure Lengend Snippet: Analysis performed at 1000× coverage on E. coli 16S rRNA (left) and 23S rRNA (right). (a) AUPRC as a function of symmetric tolerance window size (±0 to ±10 nt from known modification sites). Each line represents one tool; shaded bands indicate ±SD across biological replicates. The beige region indicates approximate 5-mer reader head context. (b) δ AUPRC heatmaps showing the change in AUPRC relative to exact-position evaluation (w = 0, vertical black line) for each tool and window size. Red indicates gains and blue indicates losses from the exact match. Stars mark the largest absolute change per tool. (c, left) Effective modification prevalence across window sizes, showing the fraction of positions counted as positive as windows expand. (c, right) Dumbbell chart comparing each tool’s AUPRC at exact position (open circles) with its peak AUPRC across all window sizes (filled circles), shown separately for 16S (blue) and 23S (red) rRNA.

Article Snippet: We polyadenylated total RNA to enable direct RNA sequencing library preparation using the E. coli poly(A) polymerase kit (New England Biolabs, cat# M0276).

Techniques: Modification