The 3’-amino and 5’- C6SSC6 disulfide modified oligonucleotide (Axolabs, Kulmbach, Germany) was dissolved in 100 mM sodium borate buffer containing 20% acetonitrile (pH 8.5) to a final concentration of 800 μM. Atto488-NHS ester (Atto-Tec, Siegen, Germany) was dissolved in anhydrous DMSO to a working concentration of 1 mM. Three molar equivalents of Atto488-NHS ester solution were added over 2 h every 15 min, following 3 h incubation at 25°C. The resulting construct was purified by EtOH precipitation and redissolved in water to a concentration of 1 mM. The C6SSC6 disulfide modified end was reduced with buffered tris(2-carboxyethyl)phosphine (TCEP, 700 times molar excess, Sigma Aldrich, Steinheim, Germany) for 2.5 h at RT. TCEP was removed by EtOH precipitation. The remaining pellet was redissolved in 50 mM sodium phosphate buffer 20% acetonitrile (pH 7) to a concentration of 800 μM. Tetramethylrhodamine-6-maleimide (Life Technologies, Darmstadt, Germany) was dissolved in anhydrous DMSO to a working concentration of 1 mM. The Tetramethylrhodamine-6-maleimide solution (1.3 equivalents) was added immediately to the oligonucleotide solution, following incubation of 2 h at 25°C. The product was purified by EtOH precipitation and high-performance liquid chromatography.
Atto 488 nhs ester
Manufactured by ATTO-TEC
Sourced in Germany
ATTO 488 NHS-ester is a fluorescent dye molecule used for labeling biomolecules such as proteins, peptides, and nucleic acids. It has an excitation maximum at 501 nm and an emission maximum at 523 nm, making it suitable for detection in the green spectral region.
Automatically generated - may contain errors
Lab products found in correlation
Resomer rg 503h, by Merck Group (2 mentions)
Atto 633 amine, by ATTO-TEC (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Surfactant p20, by GE Healthcare (1 mentions)
Monolith nt 115 system, by NanoTemper (1 mentions)
Resomer rg 752h, by Merck Group (1 mentions)
Superoxide dismutase from bovine erythrocytes, by Merck Group (1 mentions)
Cellmask green plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
Atto550nhs ester, by ATTO-TEC (1 mentions)
Polyethyleneimine pei branched mw 25 000 da, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Tetramethylrhodamine 6 maleimide, by Thermo Fisher Scientific (1 mentions)
Dipea, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
C18 column, by Agilent Technologies (1 mentions)
Dylight650 nhs ester, by Thermo Fisher Scientific (1 mentions)
Cf568 succinimidyl ester, by Merck Group (1 mentions)
Atto 643 nhs ester, by ATTO-TEC (1 mentions)
Oligo clean concentrator, by Zymo Research (1 mentions)
μ slide 8 well chamber, by Ibidi (1 mentions)
12 protocols using atto 488 nhs ester
1
Fluorescent Oligonucleotide Dual-Labeling Protocol
2
Characterizing CoV-2 Mac1 Interactions
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MST measurements followed the framework outlined by (Seidel et al., 2013 (link)). Purified CoV-2 Mac1 was labeled with Atto488 NHS-ester (ATTO-TEC) according to the manufacturer’s protocol with labeling efficiency 1:1 protein-to-dye ratio. Labeled CoV-2-Mac1 (200 nM) or a combination of labeled (100 nM) and unlabeled (400 nM) CoV-2 Mac1 was combined with 0.01–2.5 mM ADP-ribose, PARG-329, or PARG-345 in MST buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 0.05% Surfactant P20, GE Healthcare; 5% DMSO was included for drug titrations), incubated for 10–15 min at room temperature, and loaded into standard silica capillaries (NanoTemper). Microscale thermophoresis (MST) measurements were acquired on a Monolith NT.115 system (NanoTemper) at 25 °C with 10–20% LED power and 40% (ADP-ribose) or 60% (PARG-329, PARG-345) infrared excitation for 20–30 s with 5-s equilibration and recovery periods. Data were analyzed with Nano Temper analysis software and displayed with Graphpad Prism 8.
ITC measurements were performed as described above with a PEAQ-ITC instrument using a cell concentration of DMSO-matched 50 μM CoV-2 Mac1 with 3 mM drug in the syringe (25 mM HEPES, pH 7.5, 150 mM NaCl, 6% DMSO). Data were analyzed with the MicroCal PEAQ-ITC Analysis Software.
ITC measurements were performed as described above with a PEAQ-ITC instrument using a cell concentration of DMSO-matched 50 μM CoV-2 Mac1 with 3 mM drug in the syringe (25 mM HEPES, pH 7.5, 150 mM NaCl, 6% DMSO). Data were analyzed with the MicroCal PEAQ-ITC Analysis Software.
3
PLGA Polymer Labeling Protocol
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PLGA (Resomer RG 503H and Resomer RG 752H) was purchased from Sigma-Aldrich and used as received. ATTO 488–NHS ester and Atto 633 amine were purchased from ATTO-TEC GmbH (Germany) and used as stock solutions (10 mg/ml) in dimethyl sulfoxide (DMSO).
All other chemicals were purchased from Sigma-Aldrich, unless otherwise specified. All chemicals were used as received.
All other chemicals were purchased from Sigma-Aldrich, unless otherwise specified. All chemicals were used as received.
4
Characterization of PLGA-PEI Nanoparticles
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Poly(D,L-lactide-co-glycolide) (PLGA; Resomer RG 503H) was purchased from Sigma-Aldrich (Darmstadt, Germany) and used as received. Poly(ethylene imine) (PEI), branched, MW 25.000 Da was purchased from Sigma-Aldrich (Darmstadt, Germany) and used as 14 mg/mL stock solution in dimethyl sulphoxide (DMSO). Bovine Serum Albumin (BSA) and Superoxide Dismutase from bovine erythrocytes were purchased from Sigma-Aldrich (Dartmstadt, Germany) and used as received. Atto 488 NHS-ester, Atto 550 NHS-ester and Atto 633 amine were purchased from Atto-TEC GmbH (Siegen, Germany) and used as 10 mg/mL stock solution in DMSO. Fluorescently labelled PLGA was obtained as described elsewhere [16 (link)]. Lysotrack Red DND-99 and CellMask Green Plasma Membrane Stain were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and used as received. All other chemicals were purchased from Sigma Aldrich (Darmstadt, Germany) unless specified and used as received.
5
Labeling of Peptides with Fluorescent Dyes
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CTD peptides were N-terminally labeled with Atto-488, while NCoR and FMR1 peptides were purchased with an N-terminal FAM label. 0.5 mg of lyophilized CTD peptides were dissolved in 30 µL DMSO (Sigma-Aldrich). One molar equivalent of Atto-488 NHS ester (Atto-tec) and five equivalents of DIPEA (Sigma-Aldrich) was added to three equivalents of the peptide. Reactions were incubated ON at room temperature (RT) and protected from light. Labeled peptides were purified by reverse-phase HPLC over a C18 column (Agilent Technologies) using a gradient from 30 to 70% Methanol (Merck). Purified peptides were lyophilized and stored at −20 °C.
6
Fluorescent Labeling and Cellular Uptake of DNA, Complexes, and Receptor Complexes
7
Labeling E. coli Ribosomes with Fluorescent Dyes
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Sucrose gradient purified E. coli BL21 70S ribosomes were labelled as previously reported (2) using either ATTO488 NHS-ester (ATTO-TEC GmbH), or Dylight650 NHS-ester (Thermofischer Scientific) dyes. Briefly, 4.8 µM 70S ribosomes and 250 µM dye was mixed together in the reaction buffer (50 mM TrisHCl, pH 7.6, 15 mM MgCl 2, 100 mM NH4Cl and 6 mM β-mercaptoethanol) and incubated at 37 C for 30 minutes. Any precipitate was removed by centrifugation at 10000 xg for 1 minute using a tabletop centrifuge. The supernatant containing ribosomes was concentrated in a spin column (Vivaspin 6, MWCO 30 kDa) and thoroughly washed with reaction buffer to remove excess dye.
The final concentration of labelled 70S ribosome (A488) was estimated at 5.1 µM, and the ATTO488 label concentration to be 27.5 µM using a Nanodrop 1000 (Isogen), corresponding to ~5-6 labels per ribosome. The labelled 70S ribosome (D650) was estimated at 6.3 µM, and the Dylight650 label concentration at 28.7 µM using a Nanodrop 1000 (Isogen), corresponding to ~4-5 labels per ribosome. The ribosomes were then aliquoted, flash frozen in liquid nitrogen and stored at -80°C until further use.
The final concentration of labelled 70S ribosome (A488) was estimated at 5.1 µM, and the ATTO488 label concentration to be 27.5 µM using a Nanodrop 1000 (Isogen), corresponding to ~5-6 labels per ribosome. The labelled 70S ribosome (D650) was estimated at 6.3 µM, and the Dylight650 label concentration at 28.7 µM using a Nanodrop 1000 (Isogen), corresponding to ~4-5 labels per ribosome. The ribosomes were then aliquoted, flash frozen in liquid nitrogen and stored at -80°C until further use.
8
Fluorescent DNA Probe Conjugation
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In each 10 μl reaction, 2 μl of 0.5 mM 5’ amine-modified DNA probes (Integrated DNA technologies) is mixed with 1 μl of 10 mM ATTO488-NHS ester (ATTO-TEC AD 488–31), ATTO 643-NHS ester (ATTO-TEC AD 643–31) or CF568 succinimidyl ester (Sigma-Aldrich SCJ4600027) in 1x BBS (Thermo Fisher Scientific 28384) pH 8.5, and incubate at room temperature for 4 hours. Fluorophore-conjugated DNA probes were purified with Oligo Clean & Concentrator (Zymo Research D4060), and diluted to 1μM in ultrapure water, aliquoted and stored at −20C. Oligonucleotide sequences and fluorophores used in the GFP-targeting screen were listed in Supplementary Table 4 , the base-editing screens and in vivo CRISPRmap barcode readout were listed in Supplementary Table 5 .
9
Synthesis and Labeling of O6-Methylguanine
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O6-Methylguanine (O837914) was obtained from Macklin. Atto488 NHS-ester was obtained from Atto-Tec, and O-(4-(aminomethyl)-benzyl)guanine was obtained from Biosynth Carbosynth.
10
Milk Gel Imaging Protocol
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Milk fat was stained for imaging with Nile Red (Sigma Aldrich, UK) to a concentration of 2.5 μM in the milk. Stained samples were left overnight at 5°C. Milk acid gels were prepared by adding 0.25 g of Glucono-δ-Lactone (GDL)(Sigma-Aldrich, USA) to 10 mL of the pre-stained milk giving a concentration of 2.5% GDL in the milk. The 10 mL of milk with GDL was inverted by hand for 1 min, and 600 μL of milk was sampled to which 3 μL of Atto 488 NHS-Ester (Atto-Tec GmbH, Siegen, Germany), dissolved in DMSO (99.9% pure, Sigma-Aldrich) was added to give a concentration of 510 μM in the milk. This volume was then transferred to a μ-Slide 8 Well chamber (ibidi, Germany), and incubated at 35 °C for 90 min. Five gels were produced per milk sample, giving 10 gels per treatment, as milk samples were prepared in duplicate.
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