Structure and Dynamics of Triazole-Linked DNA: Biocompatibility Explained
Structure and Dynamics of Triazole-Linked DNA: Biocompatibility Explained
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DOI：
10.1002/chem.201102979
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发表时间：
2011-12-01
影响因子：
4.3
通讯作者：
Brown, Tom
中科院分区：
文献类型：
--
作者：
Dallmann, Andre;El-Sagheer, Afaf H.;Brown, Tom
标准DNA亚磷酰胺、固体载体和其他试剂从Link Technologies、应用生物系统公司和PROLIGO购买。寡核苷酸是在应用生物系统公司394自动DNA/RNA合成仪或AKTAOlyopilot Plus合成仪上合成的，使用的是标准的1.0或15.0微克分子亚磷酰胺循环，包括酸催化的去硝化、偶联、封顶和碘氧化。逐步偶联效率和总产率由自动三叔丁基阳离子电导率监测设备测定，在所有情况下均为98.0%。所有β-氰乙基亚磷酰胺单体在使用前立即溶解在浓度为0.1M的无水乙腈中。3‘-炔丙基-dT(TK)和5’-碘-dT-亚磷酰胺单体的偶联时间由标准的40 S改为480S。未修饰的寡核苷酸用反相高效液相色谱法进行纯化。采用Gilson色谱柱，乙腈-乙酸铵(0%~50%缓冲液B，30min，流速4 m L/min)，缓冲液A：0.1M醋酸铵，pH 7.0，缓冲液B：0.1M醋酸铵，pH 7.0，pH 7.0，用50%乙腈。用300 nm处的紫外吸收监测洗脱情况。寡核苷酸在ACTA净化器上用Hiprep 26/10脱盐柱脱盐，凝胶电泳法分析。
Standard DNA phosphoramidites, solid supports, and additional reagents were purchased from Link Technologies, Applied Biosystems and Proligo. Oligonucleotides were synthesized on an Applied Biosystems 394 automated DNA/RNA synthesizer or AKTAOligopilot Plus synthesizer using a standard 1.0 or 15.0 µmol phosphoramidite cycle of acid-catalyzed detritylation, coupling, capping, and iodine oxidation. Stepwise coupling efficiencies and overall yields were determined by the automated trityl cation conductivity monitoring facility and in all cases were> 98.0%. All β-cyanoethyl phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use. The coupling time for the 3’-propargyl dT (TK) and 5’-iodo dT phosphoramidite monomers was changed from the standard 40 s to 480 s.The unmodified oligonucleotides were purified by reversed-phase HPLC on a Gilson system using PLRPS column (Varian) with a gradient of acetonitrile in ammonium acetate (0% to 50% buffer B over 30 min, flow rate 4 mL/min), buffer A: 0.1 M ammonium acetate, pH 7.0, buffer B: 0.1 M ammonium acetate, pH 7.0, with 50% acetonitrile. Elution was monitored by UV absorption at 300nm. After HPLC purification, oligonucleotides were desalted on an ACTA purifier using a Hiprep 26/10 desalting column and analyzed by gel electrophoresis.