DNA strands containing 5-hydroxyuracil (U OH ) nucleobases were synthesized on an automated DNA synthesizer (NTS M-2-MX DNA/RNA synthesizer)30 (link). The DNA synthesis was carried out on a 1-μmol scale in a DMTr-on mode with ultramild deprotection phosphoramidites and reagents (Glen Research). Note that the phosphoramidite monomer of U OH deoxynucleoside was synthesized following the literature60 (link),61 (link) or purchased from Glen Research (catalog No. 10-1053). The coupling time of U OH was extended to 15 min. The products were deprotected using 28% NH3 aqueous solution at room temperature for 2–3 h and then purified and detritylated using a PolyPak II cartridge (Glen Research). Some of the U OH -containing strands were prepared by the ligation of a shorter U OH -containing strand and a natural strand by using a T4 DNA ligase. The oligonucleotides were purified by reverse-phase HPLC (Waters XBridge C18 column) or by denaturing polyacrylamide gel electrophoresis. All DNA strands were identified by MALDI-TOF or ESI-TOF mass spectrometry (Supplementary Table 2 ).
Polypak 2 cartridge
Manufactured by Glen Research
The PolyPak II cartridge is a laboratory equipment product designed for solid-phase extraction (SPE) applications. It provides a convenient and efficient way to purify, concentrate, or isolate target analytes from complex sample matrices. The PolyPak II cartridge contains a sorbent material that selectively retains the analytes of interest, allowing for their separation from interfering substances. This product is suitable for use in a variety of analytical and research applications, but a detailed description of its intended use is not available.
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6 protocols using polypak 2 cartridge
1
Synthesis and Purification of 5-Hydroxyuracil-Containing DNA
2
Synthesis and Purification of Bpy-modified DNA
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Oligodeoxynucleotides were synthesized on an Applied Biosystems 394 DNA synthesizer by standard phosphoramidite chemistry. The synthesis of the phosphoramidite derivative of Ubpy is presented in the ESI.† The DNA synthesis was carried out on a 1 μmol scale in DMTr-on mode with standard reagents purchased from Glen Research. The coupling time of the nucleosides was extended to 15 min. The products were deprotected in 25% NH3 solution at 55 °C for 8 h. The oligomers were firstly purified and detritylated using a PolyPak II cartridge (Glen Research) and further purified by reverse-phase HPLC (Waters XBridge C18 column, 0.1 M TEAA (pH 7.0)/MeCN gradient, 60 °C) (Fig. S1† ). All DNA strands were identified by MALDI-TOF mass spectrometry (see ESI† ). The amount of the oligomers was determined based on the UV absorbance at 260 nm. The molar extinction coefficients (ε260) of the bpy-modified DNA strands (L1 , L2 , and L3 ) were estimated15 by the sum of the ε260 value of the bpy group and that of corresponding unmodified oligonucleotides calculated by the nearest-neighbor method. Some of the unmodified oligonucleotides purified by HPLC were purchased from Japan Bio Services and used without further purification.
3
Synthesis and Purification of Labeled DNA
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Unlabeled, 4% 15N-labeled and 2H-labeled DNA oligonucleotides were chemically synthesized using an ABI 394 DNA/RNA synthesizer. Necessary reagents were purchased from Glen Research. Samples were purified with a Poly-Pak II cartridge (Glen Research) and dialyzed successively against water, 25 mM KCl solution and water. Prior to all experiments, DNA (except for T95–2T and 93del ) samples were heated at ∼95°C for 10 min and quickly cooled down to room temperature using cold water (termed as ‘quenching’ procedure). The samples typically contained 35 mM KCl and 10 mM potassium phosphate (KPi) (pH 7.0), supplemented with 1 mM EDTA and 0.1 mM NaN3. Guanosine was purchased from Sigma-Aldrich and preapred at 150-200 mM in 1 M KOH solution.
4
FLT3-ITD Antisense Oligonucleotide Design
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For FLT3‐ITD ASO, a series of 16‐nucleotide (nt) ASOs were designed to specifically target the repeated region of FLT3‐ITD. The ASOs have a 3‐10‐3 gapmer configuration (i.e. 3‐nt locked nucleic acid [LNA]‐modified ends flanking a central 10‐nt DNA segment) and are fully modified with phosphorothioate (PS) chemistry throughout the backbone. All FLT3‐ITD ASOs were synthesized in‐house with an ABI 394 DNA/RNA synthesizer on Glen UnySupport (Glen Research) using standard phosphoramidite chemistry. LNA phosphoramidites purchased from Sigma‐Aldrich, and phenylacetyl disulfide (ChemGenes Corporation) was used as the sulfurizing reagent. Oligonucleotide cleavage and deprotection were performed under concentrated aqueous ammonia at 55°C for 16 h. The ASOs were purified with Poly‐Pak II cartridges (Glen Research) following the manufacturer's protocol, desalted using Glen Pak 2.5 desalting column (Glen Research), and dried by lyophilization. The ASOs were characterized by JEOL SpiralTOF matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometer.
miR‐125b ASO and FAM‐conjugated ASO (FAM ASO) were synthesized by Shanghai Genepharma (Shanghai, China) as described in our previous paper.19
miR‐125b ASO and FAM‐conjugated ASO (FAM ASO) were synthesized by Shanghai Genepharma (Shanghai, China) as described in our previous paper.
5
Synthesis and Annealing of siRNA
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All siRNAs, including the model double-stranded oligodeoxyribonucleotide, were synthesized using an ASM-800 DNA/RNA synthesizer (Biosset, Novosibirsk, Russia). Each strand was synthesized separately by following the standard RNA synthesis procedures and utilizing TOM-protected monomers (Glen Research, Sterling, VA, USA). The cleavage and deprotection protocols specified by the supplier were followed. Purification of synthesized oligonucleotides was performed by IE HPLC followed by desalting by PolyPak II cartridges (Glen Research) according to standard protocols. The siRNA concentration was determined by measuring the absorbance of each strand at 260 nm and using conversion factor of 1 OD being equal to 40 μg/mL of single-stranded RNA. The siRNA strands were immediately annealed before conducting biological experiments, using Dharmacon siRNA buffer consisting of 20 mM KCl, 6 mM HEPES, 0.2 mM MgCl2 × 6H2O, and a pH of 7.5.
6
Synthesis and Characterization of Labeled Oligonucleotides
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Unlabeled and 4% site-specific 15N-labeled oligonucleotides were synthesized on an ABI 394 DNA/RNA synthesizer. Standard DNA phosphoramidites, solid supports and additional reagents were purchased from Glen Research Corporation. 15N-labeled phosphoramidites were purchased from Cambridge Isotope Laboratories. Cleavage from the solid support, deprotection and purification (by PolyPak II cartridges, Glen Research) of oligonucleotides were performed according to the manufacturer's protocols. Purified oligonucleotides were subsequently dialyzed successively in deionized water, 25 mM KCl, and again in deionized water, and freeze-dried. Oligonucleotides were dissolved in buffer comprising 20 mM potassium phosphate (KPi) (pH 5.0–9.0), 70 mM KCl and 10% D2O. DNA concentration was expressed in strand molarity. Extinction coefficient of the modified sequences was approximated to that of the unmodified sequence.
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