The thermodynamics of formation of a three-strand, DNA three-way junction complex.
The thermodynamics of formation of a three-strand, DNA three-way junction complex.
复制标题
三链 DNA 三路连接复合物形成的热力学。
DOI：
10.1021/bi00188a011
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Leontis,NB
中科院分区：
文献类型：
--
作者：
Ladbury,JE;Sturtevant,JM;Leontis,NB
材料和方法核酸合成和纯化。基于解链曲线的研究已经建立了DNA TWJ的热力学参数（每个臂中有五个碱基对; Leontis等人，1991），并表明DNA TWJ复合物的形成通过在一条链中的分支点包含两个或更多个未配对碱基而稳定。因此，在本文所述的工作中，我们在S3上掺入了两个腺苷核苷酸（因此，S3 AA）。寡核苷酸由米德兰认证试剂公司（米德兰，德克萨斯州）以10 μ 1规模使用氨基磷酸酯化学合成，并通过制备型HPLC使用离子交换色谱法（对于寡核苷酸S1和S2）或反相色谱法（对于寡核苷酸S3 AA）纯化。通过UV吸光度，使用由公开的最近邻参数计算的消光系数（Puglisi & Tinoco，1989）定量寡核苷酸浓度。计算以下摩尔消光系数（260 nm），对于SI（S '-GCCTGCCACCGC），102 480;对于S2（S'-GCGGTGCGTCCG），111 140;和对于S3 AA（5 '-CGGACGAAGCAGGC），143 340。通过在样品缓冲液中溶解之前称量冻干的寡核苷酸来独立地检查样品浓度。使用天然凝胶电泳，通过对寡核苷酸对的迁移率变化滴定进一步验证相对浓度。
MATERIALS AND METHODSNucleic Acid Synthesis and Purification. Studies based on melting curves have established thermodynamic parameters for DNA TWJs (with five base pairs in each arm; Leontis et al., 1991) and indicate that the formation of DNA TWJ complexes is stabilized by the inclusion at the branch point of two or more unpaired bases in one strand. Thus, in the work reportedhere, we incorporated twoadenosine nucleotides on S3 (hence, S3AA).Oligonucleotides were synthesized by the Midland Certified Reagent Company (Midland, TX) on a 10-µ 1 scale using phosphoramidate chemistry and were purified by preparative HPLC using ion-exchange chromatography (for oligos SI and S2) or reverse-phase chromatography (for oligo S3AA). Oligonucleotide concentrations were quantified by UV absorbance using extinction coefficients calculated from pub-lished nearest neighbor parameters (Puglisi & Tinoco, 1989). The following molar extinction coefficients (260 nm) were calculated, for SI (S'-GCCTGCCACCGC), 102 480; for S2 (S'-GCGGTGCGTCCG), 111 140; and for S3AA (5'-CGGACGAAGCAGGC), 143 340. Sample concentrations were independently checked by weighing the lyophilized oligonucleotides before dissolution in the sample buffer. The relative concentrations were further verifiedby mobility shift titration of pairs of oligonucleotides using native gel elec-