mitochondrial assay buffer  (Valiant Co Ltd)

Journal: bioRxiv

Article Title: Mitochondrial genome microhomology-mediated editing by donor DNA delivery into mitochondria in human cells

doi: 10.1101/2025.11.02.686110

Figure Lengend Snippet: (A) Sequences of the wild-type and LHON-associated mutant ND4 gene alleles and ND4 proteins (upper panel) aligned to MMEJ-L-HS and MMEJ-R-HS duplexes (lower panel). Nucleotides and amino acid residues, which differ between wild-type and mutant alleles and proteins, are highlighted in purple. Sequence within the HS rectangle corresponds to the microhomology recombination site HS. (B) DNA duplexes applied for BamHI site insertion by MMJE (lower panel) and changes introduced in the ND4 gene and ND4 protein during the mitochondrial genome editing by donor DNA delivery alone. The edited ND4 gene nucleotides and corresponding amino acids are highlighted in bold gray. Sequences in the HS-1 and HS-2 rectangles correspond to the microhomology recombination sites HS-1 and HS-2. (C) End-joining activity in mitochondrial and nuclear extracts tested with DNA duplexes MMEJ-L-HS and MMEJ-R-HS (Supplementary Table 2) as schematically represented at the top. The MMEJ product was detected by radioactive PCR applying P5 and 32 p-labeled P7 primers (highlighted in red) and separated by 10% urea PAGE. The HS sequence is shown on the panel (A). Bands above the MMEJ band correspond to the NHEJ products due to NHEJ activity from the nuclear extract (lane 3). (D) End joining activity tested in mitochondrial and nuclear extracts in presence of three DNA duplexes: MMEJ-L-HS1, MMEJ-Linker, and MMEJ-R-HS2, as schematically shown on the upper panel. The blue section in the middle of MMEJ-Linker represents the BamHI site. HS-1 and HS-2 sequences are shown on panel (B). Nucleotide G11778, which is commonly mutated in LHON, is underlined and enlarged. Nucleotides, which are different from those in the natural mitochondrial genome sequence and create the BamHI site, are in red and underlined. Autoradiography of amplicons, corresponding to MMEJ PCR product and its fragment (MMEJ BamHI Digestion Band) and separated by 10% urea PAGE, is shown. DNA Ladder represents a mix of 32 p-labelled oligonucleotides, their size is indicated at the right.

Article Snippet: 1/20 of the nuclei-containing pellet after the first centrifugation was lysed in a mitochondrial lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 10% glycerol, 2 mM EGTA, 2 mM EDTA, 0.2% Triton X-100, and 1 mM DTT) complemented with Half Protease and Phosphatase Inhibitor Cocktail (cat # 78441, ThermoFisher Scientific) by suspending the pellet in 100μl of the ice-cold mitochondrial lysis buffer and leaving the suspension on ice for 30 minutes and centrifuging it at 13,200g for 5 minutes at +4°C.

Techniques: Mutagenesis, Sequencing, Activity Assay, Labeling, Autoradiography

Journal: bioRxiv

Article Title: Mitochondrial genome microhomology-mediated editing by donor DNA delivery into mitochondria in human cells

doi: 10.1101/2025.11.02.686110

Figure Lengend Snippet: (A) Chimeric RNA-DNA oligonucleotides RMIS-Dir, RMIS-Rev, as well as Dir and Rev, designed for mitochondrial delivery and editing experiments are shown in alignment to the MMEJ-Linker duplex. Nucleotides which differ from the wild-type ND4 sequence are highlighted in gray. The BamHI site is shown by the small rectangle. Nucleotides, which differ from the wild-type sequence, are highlighted in bold. The blue hairpin structures represent RMIS. (B) Schematic representation of submitochondrial fractionation (modified from ). (C, D) In vivo import Assays. Mitochondria were purified from the HEK 293T cells transfected with Duplex 1 RMIS-Dir:RMIS-Rev (C) or with Duplex 2 RMIS-Dir:Rev (D). Total nucleic acids (RNA + DNA) were purified and separated by 10% urea-PAGE for Northern blot hybridization. Two representative blots are shown for each experiment (left and right panels), probed for oligonucleotides Dir or Rev (upper left and right panels, correspondingly) as indicated on the left of each panel. To detect possible cytosolic contamination, the probe for 5.8S ribosomal RNA was used (middle panels); the mitochondrial tRNA Thr signal (lower panel) served as a loading control and as an indicator of mitochondrial and mitoplast integrity. Mitochondria, mitoplasts and lysed mitochondria are schematically indicated above the panels, as in (B). Markers of size and hybridization controls, oligonucleotides RMIS-Dir and Dir, were loaded on the right extremity of each gel, as indicated on the top.

Article Snippet: 1/20 of the nuclei-containing pellet after the first centrifugation was lysed in a mitochondrial lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 10% glycerol, 2 mM EGTA, 2 mM EDTA, 0.2% Triton X-100, and 1 mM DTT) complemented with Half Protease and Phosphatase Inhibitor Cocktail (cat # 78441, ThermoFisher Scientific) by suspending the pellet in 100μl of the ice-cold mitochondrial lysis buffer and leaving the suspension on ice for 30 minutes and centrifuging it at 13,200g for 5 minutes at +4°C.

Techniques: Sequencing, Fractionation, Modification, In Vivo, Purification, Transfection, Northern Blot, Hybridization, Control

Journal: bioRxiv

Article Title: Mitochondrial genome microhomology-mediated editing by donor DNA delivery into mitochondria in human cells

doi: 10.1101/2025.11.02.686110

Figure Lengend Snippet: (A) Schematic representation of ss-oligonucleotides and duplexes used in the corresponding transfections of HEK 293T cells. Duplex 1 – RMIS-Dir:RMIS-Rev; Duplex 2 – RMIS-Dir:Rev; Duplex 3 – Dir:RMIS-Rev. (B) Mitochondrial genome editing efficiency in counts of reads with the edited mitochondrial sequence per one million (Counts Per Million = CPM) of reads unambiguously aligned to the mitochondrial sequence (MT:11713-11853) for each transfection is shown on the graph. Statistical significance was assessed by the two-sided permutation test with the plus-one correction and adjustment via Benjamini-Hochberg False Discovery Rate. N.S. – no statistical significance (p.adjust > 0.3). * – moderate statistical significance (p.adjust < 0.05; # 1 vs # 2 – p.adjust = 0.0036; # 1 vs # 3 – p.adjust = 0.043; # 1 vs # 4 – p.adjust = 0.0069). ** – strong statistical significance (p.adjust < 0.001).

Article Snippet: 1/20 of the nuclei-containing pellet after the first centrifugation was lysed in a mitochondrial lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 10% glycerol, 2 mM EGTA, 2 mM EDTA, 0.2% Triton X-100, and 1 mM DTT) complemented with Half Protease and Phosphatase Inhibitor Cocktail (cat # 78441, ThermoFisher Scientific) by suspending the pellet in 100μl of the ice-cold mitochondrial lysis buffer and leaving the suspension on ice for 30 minutes and centrifuging it at 13,200g for 5 minutes at +4°C.

Techniques: Transfection, Sequencing

Journal: bioRxiv

Article Title: Mitochondrial genome microhomology-mediated editing by donor DNA delivery into mitochondria in human cells

doi: 10.1101/2025.11.02.686110

Figure Lengend Snippet: (A) crRNA, wild-type and edited ND4 alleles, and their translated protein sequences, RMIS-Dir-New and RMIS-Rev-New are shown on the picture. New HS-1 and New HS-2 indicate the microhomology arms. The guide part of crRNA and corresponding sites of wild-type and edited ND4 genes are highlighted in purple. (B) Schematic representation of a timeline of cells transfections and treatments. (C) Left panel: means of counts of reads with five-, four-, or three edited nucleotides per one million (Counts Per Million = CPM) of reads unambiguously aligned to the mitochondrial sequence (MT:11643-11870) for each transfection, are shown on the 3D graph. Each transfection was conducted in biological triplicate. Right panel: schematic explanation of the transfections and treatments.

Article Snippet: 1/20 of the nuclei-containing pellet after the first centrifugation was lysed in a mitochondrial lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 10% glycerol, 2 mM EGTA, 2 mM EDTA, 0.2% Triton X-100, and 1 mM DTT) complemented with Half Protease and Phosphatase Inhibitor Cocktail (cat # 78441, ThermoFisher Scientific) by suspending the pellet in 100μl of the ice-cold mitochondrial lysis buffer and leaving the suspension on ice for 30 minutes and centrifuging it at 13,200g for 5 minutes at +4°C.

Techniques: Transfection, Sequencing

Journal: bioRxiv

Article Title: Mitochondrial genome microhomology-mediated editing by donor DNA delivery into mitochondria in human cells

doi: 10.1101/2025.11.02.686110

Figure Lengend Snippet: The process may start with RMIS removal (likely mediated by EndoG and ExoG nucleases), followed by end resection, which could be executed by mitochondrial MMEJ machinery, and 3’- overhangs formation. The next steps may involve strand invasion and FLAP removal by FEN1, followed by DNA synthesis and ligation by DNA ligase III. Newly synthesized DNA is represented by the red dashed line.

Article Snippet: 1/20 of the nuclei-containing pellet after the first centrifugation was lysed in a mitochondrial lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 10% glycerol, 2 mM EGTA, 2 mM EDTA, 0.2% Triton X-100, and 1 mM DTT) complemented with Half Protease and Phosphatase Inhibitor Cocktail (cat # 78441, ThermoFisher Scientific) by suspending the pellet in 100μl of the ice-cold mitochondrial lysis buffer and leaving the suspension on ice for 30 minutes and centrifuging it at 13,200g for 5 minutes at +4°C.

Techniques: DNA Synthesis, Ligation, Synthesized