【作者】 卢二虎；

【导师】 彭孝军；

【作者基本信息】 大连理工大学 ， 应用化学， 2005， 博士

【摘要】 三链DNA技术具有可选择性调控基因的表达、基因的定点突变、基因的修复、原位染色体的标记和基因分子诊断等作用,因此以三链DNA为背景的反基因战略提供了治疗某些疾病的“新型药物”概念。以反基因战略为背景的各种三链DNA工具无论从化学、生物学和药物学的角度都具有重大的理论及现实意义,已经成为近几年来的研究热点之一。本文以4-氯-2-(2-萘基)喹啉为嵌入团,设计合成了一类含有三链DNA嵌入剂、连接臂和荧光团的新型T·AT三链DNA荧光探针,同时设计合成了一些三链DNA双嵌入剂。 设计并合成了一系列以N,N-双-(3-氨基丙基)甲胺为连接臂,以丹磺酰胺和萘酰亚胺为荧光团的三链DNA荧光探针。其原理是荧光探针与三链DNA结合后,由于嵌入剂嵌入到三链DNA中引起淬灭团和荧光团之间距离的变化,从而造成荧光探针光谱的变化。研究发现这些化合物嵌入到三链DNA后都可明显引起三链DNA解链温度的升高,而对双链DNA解链温度几乎没有改变,显示出这类化合物对于三链DNA具有很好的选择性。设计的大多数荧光探针对于T·AT三链DNA都有明显的荧光增强。 设计并合成了一系列以N,N′-双-(3-氨基丙基)哌嗪为连接臂,以丹磺酰胺、萘酰亚胺和菁染料为荧光团的三链DNA荧光探针,使探针的柔性长链连接臂具有某种程度的刚性。研究发现,当DNA与这类化合物以5:1的摩尔比结合时,T·AT三链DNA的△T_m为26—30℃,明显高于对应的AT双链DNA的△T_m值(2℃左右),结果与N,N-双-(3-氨基丙基)甲胺连接臂的探针类似。而以N,N′-双-(3-氨基丙基)哌嗪为连接臂的探针对T·AT三链DNA则有更大的荧光增强(4—10倍)。论文提出用荧光法测量2a与三链DNA复合物的解链温度,通过和紫外吸收法的对照验证,证明荧光法是一个可靠的测量2a与三链DNA复合物解链温度的方法,并用Smart Cycler DNA扩增仪验证了荧光法与紫外吸收法的一致性。最后用AM1半经验量子化学计算模拟1a和2a的分子构型,就两个探针分子对三链DNA具有不同的荧光增强给出了解释。 依据嵌入剂嵌入到三链DNA中引起光诱导电子转移(PET)造成荧光强度变化的机理,合成了3a,3(?)2个未见报道的以乙二胺为连接臂,以丹磺酰胺、萘酰亚胺为荧光团的三链DNA荧光探针。化合物3a对于T·AT三链DNA有很明显的荧光增强,并通过测量化合物3a对pH的响应验证了PET的作用机理。 设计合成了双嵌入剂N1—N8及单嵌入剂NO,通过紫外可见光谱测定了化合蔡基喳咐嵌入型T.AT三链DNA荧光探针研究物对不同DNA解链温度的影响,同时着重考察了化合物N6的浓度对DNA的解链温度的影响。证实经过适当的设计,双嵌入剂能够对T.AT三链产生选择性的稳定作用,并且它对三链DNA的识别作用和稳定作用均要大于相应的单嵌入剂。 关键词:三链DNA,T.AT三链ONA荧光探针,蔡基哇琳,三链DNA稳定性,双嵌入剂。更多还原

【Abstract】 DNA triple-helix formation is of great potential including selective control of gene expression, site-directed mutagenesis, gene repair, in situ chromosome labelling and other molecular diagnostics, thus the antigene strategy based on triplex DNA offers new therapeutic applications. Triplex DNA tools on this basis are of great interest from the standpoint of chemistry, medicine and biology. In this dissertation, a serial of T-AT triplex DNA probes composed of triplex DNA intercalator, spacer and fluorophore were synthesized and evaluated. Some triplex DNA bis-intercalator ligands were also synthesized and evaluated.A serial of triplex DNA probes were synthesized. In the probe, N,N-bis-(3-aminopropyl)amine was used as spacer, danyl or 4-aminonaphthalimide was used as fluorophore. The fluorescence of the probe is weak because the distance between the intercalator and the fluorophore is very close in free state. Upon intercalating into a triplex DNA, however, the probe emits greater fluorescence due to the increase of the distance between the two moieties. We found that these probes were triplex selective because they could highly enhance the Tm of T-AT triplex, but hardly enhance the Tm of AT duplex. Most compounds had fluorescence enhancement to the T-AT triplex.Another serial of triplex DNA probes were synthesized, whose spacer was N,N’-bis-(3-aminopropyl)piperazine, fluorophore was danyl, 4-aminonaphthalimide and cy-7 dye respectively. This spacer is a little more rigid than N,N-bis-(3-aminopropyl)amine. The ATm was 26 — 30°C to T-AT triplex, but only about 2°C to AT duplex. Compared with N,N-bis-(3-aminopropyl)amine spacer, N,N’-bis-(3-amino propyl)piperazine spacer had bigger fluorescence enhancement. A fluorescence melting method was brought forward to measure the Tm of the complex of 2a and T-AT triplex. This method was a’dependable method proved by UV melting method. We further proved this method by measuring the Tm of the complex of 2a and AT duplex through a Smart Cycler apparatus. An explanation for the different fluorescence response to T-AT triplex between la and 2a was presented by using of semi-empirical calculation method-AM1.A novel triplex DNA fluorescent probe, which takes 4-aminonaphthalimide or danyl as reporting group, ethylene group as a spacer and a triplex-select intercalatoras recognizing group, was synthesized based on photoinduced electron transfer (PET). Fluorescence response of probes to different DNA was studied through fluorescent spectra. We found that probe 3a was very sensitive to T-AT triplex, the fluorescence response of 3a to pH also proved the PET mechanism.Some triplex DNA bis-intercalator ligands (N1-N8) and a mono-intercalator ligand (NO) were also synthesized. The Tm values were studied through the UV melting experiments. The experiments concerning the effects of different concentrations of N6 to T-AT triplex and AT duplex suggested that appropriately designed bis-intercalator ligands were better than mono-intercalator ligands in selectively enhancing the stability of T-AT triplex.更多还原