Chromatography was performed using an Agilent 1260 chromatography system (Agilent Technologies, Santa Clara, CA, USA) and DNAPac PA200 column (4 × 250 mm) from Dionex (now Thermo Scientific, Sunnyvale, CA, USA). Samples were analyzed with a linear gradient of NaClO4 in 2 mM Tris buffer at pH 8 using a flow rate of 1 mL/min. All solvents, purchased from Sigma-Aldrich (Bangalore, India), were of HPLC-grade and used after filtering through a 0.22 μm nylon filter followed by degassing. Samples were detected using a high-sensitivity flow cell (60 mm path length) in a diode array detector. As some reactions contained more than one type of monomer, each nucleotide was injected separately as a control, and the retention time of the respective peaks was noted. In some cases, more than one monomer peak eluted together (AMP and GMP) due to the specificity of column for phosphate groups and not necessarily for the nitrogenous bases. This aspect of the column chemistry did not allow for further optimization of peak elution profiles. Since this column offered the best single-nucleotide resolution, we performed qualitative analysis using this column despite the aforementioned limitation of this technique.
Dnapac pa200 column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The DNAPac PA200 column is a specialized chromatography column designed for the separation and purification of DNA and RNA molecules. It utilizes a proprietary packing material that provides high-resolution separation of nucleic acids. The column is suitable for a variety of applications, including DNA fragment analysis, plasmid purification, and oligonucleotide purification.
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Lab products found in correlation
Agilent 1260 chromatography system, by Agilent Technologies (1 mentions)
Hitrap q, by GE Healthcare (1 mentions)
Tar rna, by Horizon Discovery (1 mentions)
Hitrap, by Cytiva (1 mentions)
Quant it ribogreen rna assay, by Thermo Fisher Scientific (1 mentions)
Nano z, by Malvern Panalytical (1 mentions)
6 protocols using dnapac pa200 column
1
Ion-exchange Chromatography of Nucleotides
2
Optimized Preparation of TAR RNA for NMR Analysis
3
Single Turnover Kinetics of TDG Enzyme
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Single turnover kinetics assay under saturating enzyme conditions was carried out to determine the rate constant (Kmax) of TDG. The reaction was performed with 5 μM TDG and 0.5 μM DNA substrates at 22 °C in the reaction buffer (25 mM HEPES, pH 7.4, 0.5 mM EDTA, 0.5 mg mL–1 BSA, and 0.5 mM DTT) and was quenched at specific time points with 50% (v : v) 0.1 M NaOH and 0.01 M EDTA. The samples were further boiled for 15 min at 85 °C (5fC and U were quenched with 0.3 M piperidine and 0.03 M EDTA and incubated at 85 °C for 15 min) and then cooled down for HPLC analysis. The products and reactants from different time points were separated and quantified by anion-exchange HPLC using reported denaturing conditions with a DNAPac PA200 column (Dionex). The oligonucleotides are detected by absorbance (260 nm), and the fraction product (F) is determined from the integrated peak areas for the product strands (AP1 and AP2) and target strand (AS) using the equation: F = (AP1 + AP2)/(AP1 + AP2 + AS), and the single turnover rate constant Kmax is calculated by using the equation: F = A[1 – exp(–Kmaxt)], in which A is the fraction of the substrate converted to the product at completion and t is the reaction time.
4
Oligonucleotide Stability Assay Using PDII
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Modified oligonucleotide was added at 0.1 mg/ml to a solution of 50 mM sodium acetate (pH 6.5) and 10 mM MgCl2. Phosphodiesterase II (PDII) from bovine spleen (Worthington, cat #LS003 602) was added to the mixture at 500 mU/ml. Aliquots were analyzed every hour for 24 h by injection onto a Dionex DNAPac PA200 column (4 mm × 250 mm) at 30°C run at a flow rate of 1 ml/min with a gradient of 37–52% Buffer B over 7.5 min. Buffer A was 20 mM sodium phosphate, 15% acetonitrile, pH 11; Buffer B was Buffer A containing 1 M sodium bromide (pH 11). The area under the peak corresponding to full-length oligonucleotide was normalized to the area from the 0 h time point. First-order decay kinetics were assumed in calculation of half-lives. A control oligo-2′-deoxynucleotide, dT20 with a single 5′-terminal PS linkage, was analyzed each day. Enzyme was prepared as a stock of 2000 mU/ml, aliquoted into 1 ml tubes, and stored at –20°C. A new aliquot was used each week.
5
Lipidoid Nanoparticle siRNA Delivery
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Nanoparticles were prepared with the cationic lipid C12-200, disteroylphosphatidyl choline, cholesterol, and PEG–DMG using a spontaneous vesicle formation formulation procedure [22 (link)]. In brief, lipids were dissolved in 90% ethanol solution and mixed with siRNA solution (25 mM citrate, pH 3 ratio) at fixed speed (1:1 ratio) and diluted immediately with PBS to final 25% ethanol. The ethanol was then removed and the external buffer replaced with PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4, pH 7.5) by dialysis. The final lipid:siRNA weight ratio was ∼ 7:1. Particle size and zeta potential were determined using a Malvern Zetasizer NanoZS (Malvern, UK). siRNA content was determined by ion exchange HPLC (Agilent) assay using DNAPac Pa200 column (Dionex Corporation Dionex, 260 nm, 55°C run at 2 mL/min). siRNA entrapment efficiency was determined by the Quant-iT RiboGreen RNA assay (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. For intravenous injection, mice were anesthetized and injected through the tail vein with 0.5 mg/kg of either siCRMP2 or control siRNA (siCON) incorporated into lipidoid nanoparticles (LNPs).
6
Oligonucleotide Stability Assay with SVPD
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Modified oligonucleotide was added to a final concentration of 0.1 mg/ml to a solution of 50 mM Tris–HCl (pH 7.2) and 10 mM MgCl2. Snake venom phosphodiesterase (SVPD) (Worthington, cat #LS003926) was added to the mixture at 750 mU/ml. Aliquots were analyzed every hour for 24 h by injection onto a Dionex DNAPac PA200 column (4 mm × 250 mm) maintained at 30°C run at a flow rate of 1 ml/min with a gradient of 40–55% Buffer B over 7.5 min. Buffer A was 20 mM sodium phosphate, 15% acetonitrile, pH 11; Buffer B was Buffer A containing 1 M sodium bromide (pH 11). The area under the peak corresponding to full-length oligonucleotide was normalized to the area from the 0 h time point. First-order decay kinetics were assumed in calculation of half-lives. A control oligo-2′-deoxynucleotide, dT20 with a single 3′-terminal PS linkage, was analyzed each day. Enzyme was prepared as a stock of 1000 mU/ml, aliquoted into 1 ml tubes, and stored at –20°C. A new aliquot was used each week.
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