All DNA oligonucleotides were chemically synthesized on an ABI 394 DNA/RNA synthesizer (Applied Biosystem) at 5 or 1 μmol scale, using the standard β-cyanoethylphosphoramidite solid-phase chemistry as described elsewhere [68 (link)]. After synthesis, the oligonucleotides were detached from the support and deprotected by treating with concentrated aqueous ammonia at 55 °C for 12 h. Filtrates and washings were concentrated under reduced pressure, dissolved in water, and purified by HPLC using standard protocols. Isolated oligonucleotides have been shown to be more than 98% pure by NMR. The oligonucleotide concentration was determined spectrophotometrically by UV adsorption measurements at 90 °C using molar extinction coefficients calculated at 260 nm by the nearest-neighbor model [69 (link)]. G4s were prepared in two different phosphate-buffered solutions (pH 7.4): 10 mM Li3PO4, 10 mM KCl, 0.2 mM EDTA (K+ buffer), and 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl (PBS buffer). Samples were then heated at 90 °C for 5 min, gradually cooled to room temperature overnight, and finally incubated at 4 °C for 24 h, before data acquisition.
Abi 394 dna rna synthesizer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 394 DNA/RNA synthesizer is a laboratory instrument designed for the automated synthesis of DNA and RNA molecules. It is capable of producing various lengths of oligonucleotides, which are essential components in various scientific applications.
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6 protocols using abi 394 dna rna synthesizer
1
High-Purity Oligonucleotide Synthesis and G4 Preparation
2
Synthesis and Purification of Telomeric DNA Oligonucleotides
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DNA oligonucleotides were chemically synthesized on an ABI 394 DNA/RNA synthesizer (Applied Biosystem) at 5 or 1 μmol scale, using the standard β-cyanoethylphosphoramidite solid phase chemistry as described elsewhere (27 (link)). In particular, the following oligonucleotides were synthesized: d[(TTAGGG)4TT] and d(TTAGGGT), corresponding to two different truncations of human telomeric DNA sequence. After synthesis, the oligomers were detached from the support and deprotected by treatment with concentrated aqueous ammonia at 55°C for 17 h. The combined filtrates and washings were concentrated under reduced pressure, dissolved in H2O, and purified by high-performance liquid chromatography (HPLC) employing standard protocols. The isolated oligomers were proved to be >98% pure by NMR. The concentration of oligonucleotides was determined by UV adsorption measurements at 90°C using appropriate molar extinction coefficient values ϵ (λ = 260 nm), calculated by the nearest-neighbor model (28 (link)). All G4s were prepared in 10 mM KH2PO4 buffer containing 70 mM KCl, pH 7.0. Samples were then heated at 90°C for 5 min, gradually cooled to room temperature overnight, and finally incubated at 4°C for 24 h, before data acquisition. The G4 parallel arrangement of the 26-mer telomeric sequence (TelG4-up) was prepared and checked as previously described (29 (link),30 (link)).
3
Synthesis and Characterization of G-Quadruplex
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The DNA sequences were synthesized using standard ß-cyanoethylphosphoramidite solid phase chemistry on an ABI 394 DNA/RNA synthesizer (Applied Biosystem) at the 18). The isolated oligomer was proved to be >98% pure by NMR. In particular, the following shown that this sequence forms a dimeric G4 in 100 mM K + -containing solution, 31 with a completely different fold compared to the monomeric G4 that is formed in buffer solution containing low amounts of K + . 31, 32 Therefore, 5 mM potassium phosphate buffer containing 20 mM KCl was also used in this case.
For the studies in the absence of metal cations, oligonucleotides (12-15 µM) were prepared in 50 mM Tris buffer, and the CD spectra were recorded 10 min after each ligand addition (stepwise additions of 0.5 equiv.). All spectra were baseline corrected and analyzed using Origin
For the studies in the absence of metal cations, oligonucleotides (12-15 µM) were prepared in 50 mM Tris buffer, and the CD spectra were recorded 10 min after each ligand addition (stepwise additions of 0.5 equiv.). All spectra were baseline corrected and analyzed using Origin
4
Synthesis and Purification of Oligonucleotides
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Oligonucleotides were prepared with an Applied Biosystems (ABI) 394 DNA/RNA synthesizer. ESI-MS spectra of oligonucleotides were performed by the Shanghai Sangon Mass Spectrometry Facility. All DNA synthesis reagents were purchased from Glen Research. The oligonucleotides were then deprotected in saturated ammonium hydroxide at room temperature for 12 h and further purified by reversed-phase HPLC on a C-18 column using 0.1 M triethylamine acetate (TEAA) buffer and acetonitrile as the eluents. The collected DNA products were dried and detritylated by dissolving and incubating DNA products in 200 μl of 80% acetic acid for 20 min. The detritylated DNA product was precipitated with NaCl (3 M, 25 μl) and ethanol (600 μl) and then desalted on a Glen-Pak DNA Purification Cartridge. The synthetic process of ON-CAG conjugates was displayed in Figure 2 .
5
Synthesis and Characterization of Acrydite-Modified Oligonucleotides
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The oligonucleotides were synthesized on the ABI 394 DNA/RNA Synthesizer (Applied Biosystems, Foster City, CA, USA). Reagents for automated DNA synthesis were purchased from Glen Research Co. (Sterling, VA, USA). For the synthesis of acrydite-modified oligonucleotides, termed aptaDNA and coDNA, capping of methacrylic phosphoramidite to the 5ʹ end of oligonucleotides was also performed on the above-mentioned machine. The acrydite-modified oligonucleotides were cleaved from the support and de-protected by 28% ammonia hydroxide at 60 °C for 24 h. Sequences of aptaDNA and coDNA are shown in Table 1 .
Acrydite-modified oligonucleotides were purified by dialysis against phosphate buffered saline (PBS) and water for 6 h each. All modified oligonucleotides were characterized by matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS, microflex LRF system, Bruker Daltonics, Bremen, Germany) in the positive ion mode. Concentration of the stock solution of each oligonucleotide was determined with UV absorption of DNA at 260 nm.
Acrydite-modified oligonucleotides were purified by dialysis against phosphate buffered saline (PBS) and water for 6 h each. All modified oligonucleotides were characterized by matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS, microflex LRF system, Bruker Daltonics, Bremen, Germany) in the positive ion mode. Concentration of the stock solution of each oligonucleotide was determined with UV absorption of DNA at 260 nm.
6
Oligonucleotide Synthesis and Characterization
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Oligonucleotides were prepared with an Applied Biosystems (ABI) 394 DNA/RNA synthesizer. ESI-MS spectra of oligonucleotides were performed by the Shanghai Sangon Mass Spectrometer (Shimadzu, Japan). Transmission electron microscopy (TEM) was carried out on an H-7000 NAR transmission electron microscope (Hitachi) with a working voltage of 100 kV. Atomic force microscopy (AFM) images of samples were obtained on a Multimode 8 (Bruker, USA). The confocal fluorescence imaging studies were performed on a confocal laser scanning microscope (FV1000, Olympus, Japan).
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