【作者】 郭俊红；

【导师】 孔德明；

【作者基本信息】 南开大学 ， 分析化学， 2010， 硕士

【摘要】 三苯甲烷染料(TPM)自身由于共振去激发显示很弱的荧光。然而当它们堆积在G-四链体的两个表面G-四分体时,由于共振被限制,它们的荧光显著增强。因此TPM染料可以开发为G-四链体识别荧光探针。本文考察了五种TPM染料和G-四链体、双链和单链DNA作用后的荧光光谱和荧光共振能量转移光谱。结果表明四种TPM染料的荧光光谱可用于区分分子内G-四链体和分子间G-四链体、单链和双链DNA。TPM染料的荧光共振能量转移光谱和荧光共振能量转移滴定可把G-四链体(包括分子内G-四链体和分子间G-四链体)与单链和双链DNA区分开。此外,还发现TPM染料所带的正电荷数以及取代基的体积是影响染料和G-四链体结合稳定性的两个重要因素。此外,本论文利用无需标记的能形成G-四链体的富G寡核苷酸链和廉价的三苯甲烷染料—结晶紫(CV)开发了一种新型的钾离子检测方法。这种钾离子定量检测方法是基于某些CV/G-四链体配合物在K+和Na+中存在不同的荧光信号,且荧光信号随K+浓度改变而变化而建立起来的。根据荧光信号随K+浓度变化趋势,我们开发出了两种检测模式。一种是荧光猝灭模式,在此模式中,荧光强度随K+增加而降低,使用的寡核苷酸链是T3TT3(5’-GGGTTTGGGTGGGTTTGGG);另一种是荧光增强模式,在此模式中,荧光强度随K+增加而增强,使用的寡核苷酸链是Hum21 (5’-GGGTTAG GGTTAGGGTTAGGG)。与已有的K+检测方法相比,此方法有以下优点：如检测费用低、检测体系中允许存在大量的Na+、检测线性范围可调及具有更长的激发和发射波长等等。另外,以Ag+和Cys为输入信号,TPM染料的荧光强度为输出信号,我们利用TPM染料／G-四链体配合物设计了一种DNA IMPLICATION逻辑门。当没有输入时,TPM染料／G-四链体配合物显示很强的荧光,此时的输出信号为1。Cys的加入对TPM染料／G-四链体配合物没有影响,所以只有输入Cys时,荧光仍旧很强,输出信号也是1。然而因为Ag+能破坏G-四链体结构的形成,所以仅有输入Ag+时,荧光猝灭,输出信号是0。当同时有Ag+和Cys输入时,Cys作为一种较强的Ag+结合剂,能使Ag+从DNA中释放出来,从而荧光得到恢复,输出信号是1。与已有的DNA逻辑门相比,此逻辑门快速、可逆并且有可靠的输出信号和数字化行为(digital behavior)。基于Ag+和Cys对TPM染料／G-四链体配合物的荧光调制作用,此体系还可以用于Ag+和Cys的高特异性均相检测。更多还原

【Abstract】 Triphenylmethane (TPM) dyes normally render rather weak fluorescence due to easy vibrational deexcitation. However, when they stack onto the two external G-quartets of a G-quadruplex (especially intramolecular G-quadruplex), such vibrations will be restricted, resulting in greatly enhanced fluorescence intensities. Thus, TPM dyes may be developed as sensitive G-quadruplex fluorescent probes. Here, fluorescence spectra and energy transfer spectra of five TPM dyes in the presence of G-quadruplexes, single-or double-stranded DNAs were compared. The results show that the fluorescence spectra of four TPM dyes can be used to discriminate intramolecular G-quadruplexes from intermolecular G-quadruplexes, single-and double-stranded DNAs. The energy transfer fluorescence spectra and energy transfer fluorescence titration can be used to distinguish G-quadruplexes (including intramolecular and intermolecular G-quadruplexes) from single-and double-stranded DNAs. Positive charges and substituent size in TPM dyes may be two important factors in influencing the binding stability of the dyes and G-quadruplexes.In addition, a novel K+ detection method was reported using a label-free G-quadruplex-forming oligonucleotide and a triphenylmethane fluorescent dye crystal violet (CV). This method is based on the fluorescence difference of some CV/G-quadruplex complexes in the presence of K+ or Na+, and the fluorescence change with the variation of K+ concentration. According to the nature of the fluorescence change of C V as a function of ionic conditions, two K+ detection modes can be developed. One is a fluorescence-decreasing mode, in which T3TT3 (5’-GGGTTTGGGTGGGTTTGGG) is used, and the fluorescence of CV decreases with an increased concentration of K+. The other is a fluorescence-increasing mode, in which Hum21 (5’-GGGTTAGGGTTAGGGTTAGGG) is used, and the fluorescence of CV increases with an increased concentration of K+. Compared with some published K+ detection methods, this method has some important characteristics, such as lower cost of the test, higher concentrations of Na+ that can be tolerated, adjustable linear detection range and longer excitation and emission wavelengths. Preliminary results demonstrated that the method might be used in biological systems, for example in urine.In this paper, a DNA IMPLICATION logic gate was also constructed based on triphenylmethane (TPM) dye/G-quadruplex complexes, using Ag+ and Cys as two inputs and fluorescence intensity of TPM dye as the output signal. The formation of the TPM dye/G-quadruplex complexes rendered the dye greatly enhanced fluorescence signal and the output signal of the gate was 1. The addition of Cys had no effect on the fluorescence signal and the output still was 1. However, the addition of Ag+ instead of Cys could greatly disrupt the G-quadruplex structure, accompanied by the fluorescence decrease of the dye, rendering an output signal of 0. The addition of Cys into Ag+-quenched fluorescence system led to the recovery of the fluorescence due to the release of Ag+ from DNA by Cys, giving an output signal of 1. Compared with previously published DNA logic gates, the gate operation is rapid and reversible with reliable, nondestructive readout and excellent digital behavior. In addition, the modulation of the fluorescence of the TPM dye/G-quadruplex complexes by Ag+ or/and Cys can also be used to develop highly selective homogenous sensing methods of Ag+ and Cys.更多还原