圆二色谱法研究碱基错配对G-quadruplex稳定性的影响

通过将参与形成四链结构G-quadruplex的G(鸟嘌呤)碱基分别替换为T(胸腺嘧啶),C(胞嘧啶)和A(腺嘌呤),用圆二色光谱系统地研究了不同位置的G对G-quadruplex结构稳定性的贡献.结果表明,(1)替换G-quadruplex结构中间层的G对其稳定性的影响最大;(2)该序列及其错配序列大多数情况下在钾离子溶液中比钠离子溶液中稳定;(3)某些特定位置的G被替换为C或A后序列的熔点与原始序列接近甚至高于原始序列.

Circular dichroism study on the stability of mismatched G-quadruplex

It is well reported that human telomeric DNA sequence [AGGG(TTAGGG)_3] can form a well-defined quadruplex structure with the hydrogen bonds between guanine bases in the appearance of K~+ and Na~+. It was reported that the formation and stability of the structure was meaningful for both gene regulation and apoptosis. In this study,the replacement of guanine bases in this sequence with thymine,adenine and cytosine at different position was carried out respectively.Their ability to form G-quadruplex as well as the G-quadruplex stability has been studied by circular dichroism (CD) spectroscopy.All oligonucleotides were synthesized and purified by Saibaisheng company.The concentrations of the oligonucleotides,expressed in singlestrand molarity,were determined by UV absorption at 260 nm using a ‘nearest-neighbor’ approximation.Circular dichroism experiments were performed on a JASCO model J-810 circular dichroism spectrapolarimeter equipped with a Peltier temperature controller.All samples (10 μmol/L in 10 mmol/L tris-HCl buffer containing 100 mmol/L NaCl at pH 7) were preheated to 95℃ for 5 min and slowly cooled to room temperature prior to measurements.A 1 mm path length cell was used for all measurements, and each spectrum was an average of five scans at 5℃.The G-quadruplex solutions were recorded in the 5℃ to 95℃ temperature range,using a scan rate of 0.5 K min~ -1 and following the variation of CD absorption at its peak around 295 nm.Analysis of the thermal curves was performed by using the software origin.Van′t Hoff thermodynamic analysis was carried out for the intramolecular G-quadruplex oligonucleotides.Their thermal reversibility (superimposable heating and cooling profiles) allowed the calculation of the value of the equilibrium constant (K) assuming a two-state model.Application of the Van′t Hoff equation (lnK=-ΔH/RT+ΔS/R) yielded to the determination of the enthalpy (ΔH) and entropy (ΔS) of the process,as described elsewhere.Results show that the replacement of guanine bases in the middle of the three stacking quartets brings the biggest influence on the stability of the formed G-quadruplex. Data also show that potassium ions bring more stability than sodium ions to the formed G-quadruplex in most cases. It is very interesting to find that,when guanine base is replaced by cytosine or adenine at specific sites,the formed structure has comparable or even higher stability as the original sequence.