Journal: Nucleic Acids Research

Article Title: High-throughput methods enabling random duplications, deletions, or nucleotide-constrained mutagenesis of entire DNA motifs

doi: 10.1093/nar/gkag236

Figure Lengend Snippet: RADDIM creates random duplications or deletions at one random position within a circular DNA molecule. ( A ) Illustration of the RADDIM workflow when starting from a plasmid template. Illustration created with BioRender.com . ( B ) An ExoChase-treated pUC19 plasmid (Fig. ) containing a CcdB toxin gene was nicked on the forward DNA-strand by the site-specific nickase Nt.BbvCI. The resulting double-nicked plasmids were incubated with Bst DNAP, with or without either the 5′–3′ single-strand-specific RecJ exonuclease, or the 3′–5′ single-strand-specific Thermolabile Exonuclease I, to determine if single-strand exonucleases could remove unwanted DNA-amplifications during a convergent nick-translation reaction. ( C ) Characterization of the DNA-ends that were created by a convergent nick-translation reaction using Bst DNAP and either RecJ or Thermolabile Exo I (Fig. ). ConNickTra linearized plasmids were purified and T7 DNAP was used to repair all DNA-ends. Next, T4 DNA ligase was used to re-circularize the linear plasmids, which were transformed into CcdB-sensitive E. coli cells. This selected for plasmids with a mutation in the ccdB toxin gene. The ccdB gene of 40 successfully sequenced plasmids from the RecJ library ( n = 40), and 39 successfully sequenced plasmids from the Thermolabile Exo I library ( n = 39) were sequenced by single-colony Sanger sequencing. ( D ) A synthetic 196 bp dsDNA fragment was incubated together with Bst DNAP alone, and/or Thermolabile Exo I, for 3 h with or without 1 mM MnCl 2 , to evaluate if manganese could reprogram the Bst DNAP and/or ExoI to degrade the ends of linear DNA molecules. ( E ) A pUC19 plasmid containing a CcdB toxin gene was randomly linearized by a ConNickTra reaction using Bst DNAP and ExoI, followed by an addition of 1 mM MnCl 2 for 10–30 min. After a T7 DNAP-mediated DNA end-repair, and a T4 DNA ligase-mediated re-circularization, plasmids were transformed into CcdB-sensitive E. coli cells. ( F ) The ccdB gene of 25 successfully sequenced plasmids from the 10 min library ( n = 25), 21 plasmids from the 20 min library ( n = 21), and 22 successfully sequenced plasmids from the 30 min library ( n = 22) were analyzed by single-colony Sanger sequencing.

Article Snippet: The RADDIM PacBio Revio deep sequencing data analysis and the Python scripts used to calculate the theoretical mutational landscapes possible with NSM are available from the Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain) GitHub for this project ( https://github.com/biosustain/raddim ) and on Zenodo ( https://doi.org/10.5281/zenodo.18863538 ).

Techniques: Plasmid Preparation, Incubation, Nick Translation, Purification, Transformation Assay, Mutagenesis, Sequencing

Journal: Nucleic Acids Research

Article Title: High-throughput methods enabling random duplications, deletions, or nucleotide-constrained mutagenesis of entire DNA motifs

doi: 10.1093/nar/gkag236

Figure Lengend Snippet: RADDIM allows for in-frame and multi-residue InDels enabling functional protein structure modifications. ( A ) Illustration of an alternative RADDIM workflow to insert random DNA sequences into a RADDIM library by ligating a random DNA sequence oligo library to ConNickTra linearized plasmids, followed by a T7 DNAP-mediated DNA-end-repair/fill-in. Illustration created with BioRender.com . ( B ) Representative β-lactamase compensatory mutations able to restore phenotypic ampicillin resistance of the enzymatically inactivated (A40P and R41W) TEM-1 protein, superimposed onto the wild-type TEM-1 protein structure (PDB: 1ZG4). Red spheres = original inactivating mutations (A40P and R41W), Green spheres = compensatory AA substitutions. Purple marking = site of multi-residue compensatory deletion. Brown marking = site of multi-residue compensatory insertions.

Article Snippet: The RADDIM PacBio Revio deep sequencing data analysis and the Python scripts used to calculate the theoretical mutational landscapes possible with NSM are available from the Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain) GitHub for this project ( https://github.com/biosustain/raddim ) and on Zenodo ( https://doi.org/10.5281/zenodo.18863538 ).

Techniques: Residue, Functional Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: High-throughput methods enabling random duplications, deletions, or nucleotide-constrained mutagenesis of entire DNA motifs

doi: 10.1093/nar/gkag236

Figure Lengend Snippet: Deep sequencing confirms the diversity of RADDIM-generated InDel libraries. ( A ) Size distribution of insertions and deletions across a RADDIM plasmid library and the location of all variants (insertions and deletions) that are 1 nt and >1 nt in length. InDels are shown by their start position in the 5′–3′ direction in the plasmid sequence. Positive values represent insertions and negative values represent deletions. CAT = chloramphenicol acetyltransferase, tCYC1 = transcriptional terminator of iso-1-cytochrome c from S. cerevisiae , ori = pUC19 origin-of-replication, BLA* = inactivated (A40P and R41W) β-lactamase (TEM-1), CcdB = bacterial DNA gyrase toxin, CcdA* = inactivated cognate immunity protein of CcdB. ( B ) Illustration of the plasmid linearization mechanisms attained by combining the ExoChase and ConNickTra methods, enabling random and singular double-stranded DNA-breaks to be enriched within only one half of a plasmid molecule, down-stream of the site-specific DNA-nick. Illustration created with BioRender.com . ( C ) Quantification of all identified deletions ranging from 1 to 30 nt in length. ( D ) Quantification of all identified insertions ranging from 1 to 30 nt in length. ( E ) The number of identified mismatches for all insertions ranging from 2 to 30 nt in length.

Article Snippet: The RADDIM PacBio Revio deep sequencing data analysis and the Python scripts used to calculate the theoretical mutational landscapes possible with NSM are available from the Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain) GitHub for this project ( https://github.com/biosustain/raddim ) and on Zenodo ( https://doi.org/10.5281/zenodo.18863538 ).

Techniques: Sequencing, Generated, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: High-throughput methods enabling random duplications, deletions, or nucleotide-constrained mutagenesis of entire DNA motifs

doi: 10.1093/nar/gkag236

Figure Lengend Snippet: RADDIM enables a random duplication or deletion of entire regulatory DNA motifs. ( A ) Illustration of the last steps in the RADDIM workflow when starting from a linear PCR-product (Fig. ). Illustration created with BioRender.com . Relative mNeonGreen fluorescent protein expression by S. cerevisiae cells transformed with RADDIM-mutated ( B ) pACT1 ( n = 90) or ( C ) pTEF1 promoter variants ( n = 86) following a FACS of top 1% of fluorescent cells. ( D ) Relative mNeonGreen fluorescent protein expression by reconstituted pACT1 and pTEF1 promoter variants ( n = 3). Statistical significance was calculated by two-way ANOVA with ns: P > 0.05, *: P ≤ 0.05, **: P ≤ 0.005, ***: P ≤ 0.0005, and ****: P ≤ 0.0001. ( E) Relative mNeonGreen fluorescent protein expression by wild-type pACT1 and pTEF1 promoters ( n = 3). Statistical significance was calculated by unpaired t -test with ns: P > 0.05 and *: P ≤ 0.0001.

Article Snippet: The RADDIM PacBio Revio deep sequencing data analysis and the Python scripts used to calculate the theoretical mutational landscapes possible with NSM are available from the Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain) GitHub for this project ( https://github.com/biosustain/raddim ) and on Zenodo ( https://doi.org/10.5281/zenodo.18863538 ).

Techniques: Expressing, Transformation Assay

Journal: Science Advances

Article Title: Pulmonary neuroendocrine cell–derived exosomes regulate iron homeostasis and oxidative stress in lung neurons

doi: 10.1126/sciadv.ady2696

Figure Lengend Snippet: ( A ) Experimental schematic showing neuronal treatment with nicotine-exposed iPNEC-derived exosomes, with or without ferristatin-II, an iron uptake inhibitor. ( B to D ) Bar graphs depict ferritin levels, ATP content, and GSH/GSSG ratios, respectively. ( E and F ) Outline showing immunocapture of SYN + exosomes from ex vivo nicotine-treated mouse lung cultures, followed by neuronal exposure with or without ferristatin-II. ( G to I ) Data indicate ferristatin-II treatment decreases ferritin accumulation, increases ATP production, and raises GSH/GSSG ratios, reflecting reduced oxidative stress. ( J ) Neuronal cells, including those with TFR1 KD were treated with immunocaptured PNEC-derived exosomes. ( K ) Immunoblot verifies efficient TFR1 KD. ( L to N ) Bar graphs show reduced ferritin, elevated ATP, and increased GSH/GSSG ratios in TFR1-deficient neurons, confirming oxidative stress reduction. ( O ) Schematic illustrating neuronal exposure to exosomes from control iPNECs, GW4869-treated iPNECs (exosome biogenesis inhibitor), or iPNECs with TF KD. ( P and Q ) Immunoblots confirm GW4869 and TF KD effects on exosome protein content. ( R to U ) Quantification reveals reduced exosome release and mitigated ferritin, ATP, and oxidative stress markers in neurons treated with modified iPNEC exosomes. ( V ) Expression of Snca and NeuN in lungs of control and P301S tau transgenic mice, with ( W ) quantified Snca expression. ( X ) Spatial transcriptomics [STARmap PLUS ] indicate enhanced neuronal vulnerability and neurodegenerative gene signatures in P301S tau transgenic mouse brains. In (B) to (D), (G) to (I), (L) to (N), (R) to (U), and (W), data are presented as means ± SEM; n = 3 independent biological replicates. Technical replicates were averaged within each experiment. Statistics: two-tailed unpaired Student’s t test for two-group comparisons, and one-way ANOVA followed by Tukey’s multiple-comparisons test for comparisons involving more than two groups. Significance levels: * P < 0.05, ** P < 0.01.

Article Snippet: The spatial transcriptomics of control and AD mouse brain were analyzed via STARmap PLUS sequencing data (Single Cell Portal of Broad Institute, Study# SCP1375) and Zenodo (DOI: 10.5281/zenodo.7332091 ) ( ).

Techniques: Derivative Assay, Ex Vivo, Western Blot, Control, Modification, Expressing, Transgenic Assay, Spatial Transcriptomics, Two Tailed Test