10x t4 ligase buffer  (New England Biolabs)

Journal: bioRxiv

Article Title: Structural and genetic dissection of RNA-guided gene repression by TnpB-derived transcription factors

doi: 10.64898/2026.09.22.753285

Figure Lengend Snippet: a, Schematic of the gRNA and target DNA substrate used for cryo-EM analysis, highlighting the TAM and 9-bp target region (see Methods for substrate design). Regions not resolved in the final reconstruction and model are shown in grey. TS, target strand; NTS, non-target strand. b, Cryo-EM density map of the TldR–gRNA–DNA ternary complex. c, Domain organization of Efa 1 TldR. The Lid motif and CTD (grey) were not resolved in the cryo-EM structure. d, Atomic model of the TldR–gRNA–DNA ternary complex in two views, colored as in a–c . e, Structure of the gRNA and target DNA, with individual RNA elements annotated. PK, pseudoknot. f, Secondary structure representation of the gRNA and target DNA, depicted as in e .

Article Snippet: DNA contamination and 5′-pyrophosphate groups were removed in a single reaction combining RppH (NEB) and TURBO DNase (Thermo Fisher), with SUPERase-In RNase Inhibitor (Thermo Fisher) included to protect the RNA, at 37 °C for 30 min. RNA ends were then repaired with T4 PNK (NEB) in 1x T4 DNA ligase buffer (NEB) for 30 min at 37 °C.

Techniques: Cryo-Electron Microscopy, Analysis

Journal: bioRxiv

Article Title: Structural and genetic dissection of RNA-guided gene repression by TnpB-derived transcription factors

doi: 10.64898/2026.09.22.753285

Figure Lengend Snippet: TldR proteins arose repeatedly from the evolutionary repurposing of ancestral, transposon-encoded TnpB nucleases (left), retaining RNA-guided DNA binding while losing DNA cleavage activity through progressive deterioration of the RuvC active site. In their domesticated form, TldRs are guided by a short guide RNA sequence to target sites positioned at or downstream of the promoter, blocking either RNA polymerase binding or its subsequent elongation, and thereby repressing transcription. In E. faecalis , this mechanism directly represses expression of a specific oppA T gene encoding a substrate-binding component of the oligopeptide permease (Opp) transporter. Because bacterial genomes often encode multiple distinct oppA homologs, repression of one variant enables alternative substrate-binding proteins to associate with the shared OppBCDF transporter, effectively shifting the spectrum of imported peptides. This regulatory logic likely provides a dynamic mechanism for tuning transporter composition in response to environmental conditions, allowing bacteria to adapt nutrient uptake and associated physiological processes to changing ecological or host-associated contexts. The native signal(s) that induce TldR expression remain unknown.

Article Snippet: DNA contamination and 5′-pyrophosphate groups were removed in a single reaction combining RppH (NEB) and TURBO DNase (Thermo Fisher), with SUPERase-In RNase Inhibitor (Thermo Fisher) included to protect the RNA, at 37 °C for 30 min. RNA ends were then repaired with T4 PNK (NEB) in 1x T4 DNA ligase buffer (NEB) for 30 min at 37 °C.

Techniques: Binding Assay, Activity Assay, Sequencing, Blocking Assay, Expressing, Variant Assay, Bacteria

Journal: Cell systems

Article Title: Capturing the dynamics of STAT6 macrophage polarization using bioluminescence temporal signatures

doi: 10.1016/j.cels.2026.101643

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: T4 DNA Ligase Buffer (10X) , NEB , B0202S.

Techniques: Recombinant, Plasmid Preparation, Purification, Staining, Yeast, Cell-Signaling, DNA Purification, Modification, Saline, Cell Culture, Transfection, Sandwich ELISA, Bicinchoninic Acid Protein Assay, Reverse Transcription, SYBR Green Assay, Lysis, Software, Spectrophotometry, Tissue, Blocking Assay, Incubator, Gel Electrophoresis, Imaging, Real-time Polymerase Chain Reaction, Laser-Scanning Microscopy