Journal:

Article Title: Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe

doi: 10.1038/nprot.2007.308

Figure Lengend Snippet: Sample data of R5 labeling and purification. (a) The sequence and secondary structure of the CS model DNA. The numbering of the phosphates is shown. (b) An example of anion-exchange HPLC results. The black trace is the 260-nm absorbance of a crude CS DNA strand with a phosphorothioate at position 4, and the red trace is the corresponding product after R5 labeling. The splitting in the unlabeled DNA is due to diastereomer separation. The labeled DNA elutes earlier than the unlabeled oligonucleotide due to the loss of a negative charge upon R5 attachment. The labeling efficiency is 94% based on the areas under respective peaks. The data shown were obtained using a PA-100 column with a flow rate of 2 ml min−1, and the linear gradient used is shown in the inset table. Variation of this gradient has been used for DNA and RNA ranging from 12 to 44 nt long. For example, for a 44-nt DNA, the gradient is modified to Step 2: 0–20% B in 4 ml; Step 3: 20–30% B in 24 ml. (c) An example of a mass spectrum of a CS DNA strand with R5 attached at position 2. (d) An example of a 20% polyacrylamide denaturing gel for examining R5 labeling. The label is attached at position 3 of a CS DNA strand. Lane 1: DNA strand without modification; Lane 2: DNA strand containing one phosphorothioate; and Lane 3: DNA strand with one R5 attached.

Article Snippet: REAGENTS list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Acetone, 99.5% (Fisher Scientific, cat. no. A929-1) Acetonitrile, 99.8% (Malinckrodt Chemicals, cat. no. 2856-10) Glycerol (molecular biology grade, Z 99.9%; Sigma-Aldrich, cat. no. G5516) Hydrochloric acid (HCl, molecular biology grade; Sigma, cat. no. H1758) 1-Oxyl-2,2,5,5-tetramethyl-3(methanesulfonyloxymethyl)pyrroline (compound 1 , ) (Toronto Research Chemicals, cat. no. O872400) 3-Iodomethyl-1-oxyl-2,2,5,5-tetramethylpyrroline (compound 2 , ) (Toronto Research Chemicals, cat. no. I709500) (see REAGENT SETUP) 2-( N -Morpholino)-ethanesulfonic acid (MES, free acid, 99.5 %; (Calbiochem, cat. no. 475893) (see REAGENT SETUP) Sodium perchlorate, 99+% (Acros Organics, cat. no. 197120010) Sodium chloride (molecular biology grade, 99.9%; Calbiochem, cat. no. 567441) Sodium iodide (NaI, 99.999%; Acros, cat. no. 212680100) 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris, molecular biology grade, 99.8%, EMD Chemicals, cat. no. 9230) (see REAGENT SETUP) MOPS, 499.5% (Sigma, cat. no. M5162) (see REAGENT SETUP) EDTA, 99.995% (Aldrich, cat. no. 431788) (see REAGENT SETUP) Sodium hydroxide (NaOH pellets, 499.998%; Sigma-Aldrich, cat. no. 480878) Sterile H 2 O (see REAGENT SETUP) Anion-exchange HPLC buffer A (see REAGENT SETUP) Anion-exchange HPLC buffer B (see REAGENT SETUP) EQUIPMENT list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Bench-model Freeze-Dryer (FTS Systems, model number Flexi-Dry MP #FD-3-85A-MP) SpeedVac (Labconco, CentriVap model 78100-00) equipped with a self-cleaning dry vacuum system (Welch Vacuum, model 2025) Anion-exchange HPLC column (Dionex PA-100, 250 × 4 mm 2 ; Dionex) G-25 Sephadex column (22.5 × 1.2 cm 2 ) Quartz capillary, OD 1.8 mm, ID 1.5 mm (Vitrocom, Inc. Mountain Lakes, NJ, cat. no. CV1518Q) HPLC system (Amersham Bioscience) Bruker ELEXSYS E580 X-band pulse spectrometer (see EQUIPMENT SETUP) REAGENT SETUP Oligonucleotides with site-specific phosphorothioate substitutions By convention, each phosphate ‘belongs’ to the ribose that is on the 3′-side of the P atom( ).

Techniques: Labeling, Purification, Sequencing, Modification

Journal:

Article Title: Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe

doi: 10.1038/nprot.2007.308

Figure Lengend Snippet: Troubleshooting table.

Article Snippet: REAGENTS list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Acetone, 99.5% (Fisher Scientific, cat. no. A929-1) Acetonitrile, 99.8% (Malinckrodt Chemicals, cat. no. 2856-10) Glycerol (molecular biology grade, Z 99.9%; Sigma-Aldrich, cat. no. G5516) Hydrochloric acid (HCl, molecular biology grade; Sigma, cat. no. H1758) 1-Oxyl-2,2,5,5-tetramethyl-3(methanesulfonyloxymethyl)pyrroline (compound 1 , ) (Toronto Research Chemicals, cat. no. O872400) 3-Iodomethyl-1-oxyl-2,2,5,5-tetramethylpyrroline (compound 2 , ) (Toronto Research Chemicals, cat. no. I709500) (see REAGENT SETUP) 2-( N -Morpholino)-ethanesulfonic acid (MES, free acid, 99.5 %; (Calbiochem, cat. no. 475893) (see REAGENT SETUP) Sodium perchlorate, 99+% (Acros Organics, cat. no. 197120010) Sodium chloride (molecular biology grade, 99.9%; Calbiochem, cat. no. 567441) Sodium iodide (NaI, 99.999%; Acros, cat. no. 212680100) 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris, molecular biology grade, 99.8%, EMD Chemicals, cat. no. 9230) (see REAGENT SETUP) MOPS, 499.5% (Sigma, cat. no. M5162) (see REAGENT SETUP) EDTA, 99.995% (Aldrich, cat. no. 431788) (see REAGENT SETUP) Sodium hydroxide (NaOH pellets, 499.998%; Sigma-Aldrich, cat. no. 480878) Sterile H 2 O (see REAGENT SETUP) Anion-exchange HPLC buffer A (see REAGENT SETUP) Anion-exchange HPLC buffer B (see REAGENT SETUP) EQUIPMENT list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Bench-model Freeze-Dryer (FTS Systems, model number Flexi-Dry MP #FD-3-85A-MP) SpeedVac (Labconco, CentriVap model 78100-00) equipped with a self-cleaning dry vacuum system (Welch Vacuum, model 2025) Anion-exchange HPLC column (Dionex PA-100, 250 × 4 mm 2 ; Dionex) G-25 Sephadex column (22.5 × 1.2 cm 2 ) Quartz capillary, OD 1.8 mm, ID 1.5 mm (Vitrocom, Inc. Mountain Lakes, NJ, cat. no. CV1518Q) HPLC system (Amersham Bioscience) Bruker ELEXSYS E580 X-band pulse spectrometer (see EQUIPMENT SETUP) REAGENT SETUP Oligonucleotides with site-specific phosphorothioate substitutions By convention, each phosphate ‘belongs’ to the ribose that is on the 3′-side of the P atom( ).

Techniques: Labeling, Gentle, Incubation, Nucleic Acid Electrophoresis, Mass Spectrometry, Concentration Assay, Solvent, Modification