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Recognition of the length of double stranded DNA by a bis-intercalator and its chromatographic application

双嵌入剂对双链DNA长度的识别及其色谱应用

基本信息

批准号：
06805074
负责人：
KAZUTA Yasuaki
金额：
$ 1.15万
依托单位：
Kyushu Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Molecular biology relies heavily on how well and quickly nucleic acids can be separated and analyzed.One of the most commonly used techniques of nucleic acid separation is gel electophoresis. If gel electrophoresis is replaced by high performance liquid chromatography (HPLC) in molecular biology, it will enable recovery of nucleic acids in a shorter period of time. Separation by gel permeation HPLC was limited to natural DNA fragments (double stranded DNA) , whereas separation by reversed phase HPLC was widely used for synthetic oligonucleotides (single stranded DNA) . DNA fragments with a difference in hundreds in base pairs have been separated in gel permeation HPLC on appropriate columns connected in tandem, but this method is too time-consuming.To solve this problem, we developed a novel method for the separation of double stranded DNA fragments by bis-intercalators. In order for bis-intercalators to bis-intercalate into DNA,a minimum length of double stranded DNA is required. If D … More NA is shorter than this length the bisacridine-intercalator binds to DNA by the mono-intercalation mode. In other words, the binding mode and affinity of the bis-intercalator for DNA varies depending on the length of DNA.We attempted to exploit this difference in the affinity of the bis-intercalator for the length of DNA.We synthesized a bis-intercalator carrying a connector which separates the two intercalator parts by 26A,equivalent to 8 bp of DNA.We also synthesized four self-complementary 6-, 8-, 12-, and 16-meric oligonucleotides.The bis-acridinyl derivative could bind to calf thymus DNA with K= (1.7<plus-minus>0.3) x10^5M^<-1> even at high salt concentration (0.1M NaCl) and cover the area of nearly eight base pairs. The effect of the bis-acridine derivative on the thermal denaturation of double stranded oligonucleotides was then studied. The bis-acridinyl derivative shifted the T_m of DNA to higher temperature, but its effect, as measured by DELTAT_m, was much more pronounced for the 8-mer or longer double stranded DNA than for the shorter counterparts. In other words, the intercalator bound the 6-mer by the mono-intercalation mode, while it bound the 8-mer or longer oligonucleotides by the bis-intercalation mode.These idea suggest that this bis-acridine derivative can discriminate double stranded DNA according to its length. By taking advantage of this characteristic precise separation of double stranded DNA of various lengths will be achieved chromatographically, once bis-intercalator is immobilized on an inert gel. Such an undertaking is now under way in our laboratory. Less

分子生物学在很大程度上依赖于核酸的分离和分析的好坏和速度。最常用的核酸分离技术之一是凝胶电泳法。如果在分子生物学中用高效液相色谱(HPLC)取代凝胶电泳法，将能够在更短的时间内回收核酸。凝胶渗透高效液相色谱分离仅限于天然DNA片段(双链DNA)，而反相高效液相色谱法广泛用于合成寡核苷酸(单链DNA)的分离。针对凝胶渗透法在串联柱上分离出碱基对相差数百个碱基对的DNA片段的问题，提出了一种利用双嵌入器分离双链DNA片段的新方法。为了使双嵌入体插入DNA，至少需要双链DNA的长度。如果D…当NA长度大于此长度时，双吖啶类插层剂以单嵌方式与DNA结合。我们合成了一种双嵌入剂，它与DNA的结合方式和亲和力随DNA长度的不同而不同。我们合成了一种带有连接体的双嵌入剂，它将DNA的两个嵌入体部分隔开26A，相当于8个DNA碱基。我们还合成了四个自互补的6-，8-，12-和16-Meric寡核苷酸。双吖啶衍生物与小牛胸腺DNA的结合系数K=(1.7&lt；正负&gt；0.3)x105m^&lt；-1&gt；即使在高盐浓度(0.1M氯化钠)下也是如此，覆盖了近8个碱基对的区域。在此基础上，研究了双吖啶类化合物对双链寡核苷酸热变性的影响。双吖啶基衍生物使DNA的T_m向更高的温度移动，但DELTAT_m测量结果表明，它对8聚体或更长的双链DNA的影响比对较短的双链DNA更明显。也就是说，该嵌入剂以单嵌插方式结合6-聚体，而以双嵌插方式结合8-聚体或更长的寡核苷酸，这表明这种双吖啶类化合物可以根据DNA的长度区分双链DNA。利用这一特性，一旦将双嵌入器固定在惰性凝胶上，就可以实现对不同长度的双链DNA的色谱精确分离。这项工作目前正在我们的实验室进行。较少