Borovok Natalia, Molotsky Tatiana, Ghabboun Jamal, Porath Danny, Kotlyar Alexander
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Aviv 69978, Israel.
Anal Biochem. 2008 Mar 1;374(1):71-8. doi: 10.1016/j.ab.2007.10.017. Epub 2007 Oct 22.
Here we describe a novel and efficient procedure for preparation of long uniform G4-DNA wires. The procedure includes (i) enzymatic synthesis of double-stranded DNA molecules consisting of long (up to 10,000 bases), continuous G strands and chains of complementary (dC)20-oligonucleotides, poly(dG)-n(dC)20; (ii) size exclusion HPLC separation of the G strands from the (dC)20 oligonucleotides in 0.1M NaOH; and (iii) folding of the purified G strands into G4-DNA structures by lowering the pH to 7.0. We show by atomic force microscopy (AFM) that the preparation procedure yielded G4-DNA wires with a uniform morphology and a narrow length distribution. The correlation between the total amount of nucleotides in the G strands and the contour length of the G4-DNA molecules estimated by AFM suggests monomolecular folding of the G strands into quadruplex structures. The folding takes place either in the presence or in the absence of stabilizing ions (K+ or Na+). The addition of these cations leads to a dramatic change in the circular dichroism spectrum of the G4-DNA.
在此,我们描述了一种制备长且均匀的G4-DNA链的新颖且高效的方法。该方法包括:(i)酶促合成由长(长达10,000个碱基)的连续G链和互补的(dC)20寡核苷酸链组成的双链DNA分子,即聚(dG)-n(dC)20;(ii)在0.1M NaOH中通过尺寸排阻高效液相色谱法从(dC)20寡核苷酸中分离G链;(iii)通过将pH值降至7.0,将纯化的G链折叠成G4-DNA结构。我们通过原子力显微镜(AFM)表明,该制备方法产生了具有均匀形态和窄长度分布的G4-DNA链。G链中核苷酸的总量与通过AFM估计的G4-DNA分子的轮廓长度之间的相关性表明,G链单分子折叠成四链体结构。折叠过程在有或没有稳定离子(K +或Na +)的情况下均可发生。添加这些阳离子会导致G4-DNA的圆二色光谱发生显著变化。