Journal: Scientific Reports

Article Title: Directed mutagenesis of large multi-subunit protein complexes by plasmid sub-fragmentation

doi: 10.1038/s41598-026-53234-8

Figure Lengend Snippet: Principles of the plasmid sub-fragmentation approach. ( a ) Linearisation by the Phusion plus DNA polymerase of the pUC19 vector, excluding the gene lacZα and the fragment n of interest, which encodes for a targeted region of Ec CI. Forward and reverse primers are represented by arrows. ( b ) CI fragment n including 15 nt complementary to the linearised product pUC19 ΔLacZa for ( c ) Gibson assembly creating the new construct pUC CI-n. The pUC CI-n construct is used for ( d ) site-directed mutagenesis, with point mutation represented by a black dot. Linearisation primers, represented by pink arrows, are designed to be 20–30 nt long, together with a 15 nt sequence complementary to both end of the ( e ) pBAD nuo linearised product (blue and red). ( f ) Linearised and the mutated fragment of pBAD nuo are incubated together for Gibson assembly, resulting in ( g ) the reassembled pBAD nuo construct, with the substitutions.

Article Snippet: When placed back to the pBAD nuo plasmid, the substitution was first confirmed to be present within the inserted fragment, followed by whole plasmid sequencing (Macrogen Europe).

Techniques: Plasmid Preparation, Construct, Mutagenesis, Sequencing, Incubation

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkag405

Figure Lengend Snippet: Improvement of in vitro transcription conditions for synthesizing precise precursor linear RNAs. ( A ) Sanger sequencing of 3′ ends of the linear RNA transcribed from unmodified DNA templates showed heterogeneity. ( B ) Schematic of amplification of DNA templates using reverse primer with two 2′-O-methyl RNA nucleosides at 3′ end to synthesize transcripts with homogeneous ends. ( C ) Sanger sequencing revealed homogenous ends of RNAs transcribed from modified DNA templates. ( D ) RNAfold modeled structures showed that addition of dinucleotides CC and CU at the 3′ends of linear precursor drastically impact the structure of the downstream circRNAs while the addition of G/T/C had no influence.

Article Snippet: Plasmid sequences were confirmed by whole plasmid sequencing (Plasmidsaurus).

Techniques: In Vitro, Sequencing, Amplification, Modification

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: Plasmid sequences were confirmed by whole plasmid sequencing (Plasmidsaurus).

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: Plasmid sequences were confirmed by whole plasmid sequencing (Plasmidsaurus).

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