ANALYSIS OF INTERACTION BETWEEN DNA STRANDS IN TRIPLE HELIX USING FLUORESCENCE CROSS CORRELATION SPECTROSCOPY.

本研究的目的是用荧光互相关光谱(FCCS)的方法来阐明DNA三螺旋中DNA链之间的相互作用特征。利用荧光相关光谱(FCS)中的自相关分析来监测杂交打开了核酸研究的新的敏感程度，但靶链的质量比必须超过标记链的8倍。本文尝试使用双色相关方法-荧光互相关光谱，来检测相同长度的双链和单链之间的相互作用。材料和方法Cy5-和罗丹明绿(RG)标记的40碱基长度的DNA，以20碱基、40碱基和对照的浓度分别为2×10~(-8)M、10~(-9)M和10~(-8)M进行测定。FCS检测采用ConfoCor 2(Zeiss)，测量时间为40s。结果两条链(正链和负链)的平移扩散时间相差不大，因为每条链的长度相等，因此用自相关方法(FCS)检测杂交产物(双链)非常困难。然而，通过计算Cy5-和RG之间的FCC的互相关信号，在没有任何物理分离方法的情况下，在均相溶液中检测到杂交过程。用方程计算出40bp的双链DNA得率约为50%，20bp的双链DNA得率约为45%。另一方面，在40个碱基和20个碱基之间杂交的情况下，由于不包含互补序列，因此没有检测到互相关信号。我们现在尝试用新的FCCS方法来检测溶液中三链DNA的形成。

The purpose of this research is to clarify the characteristic interaction between DNA strands in triple DNA helix by using fluorescence cross correlation spectroscopy (FCCS) method.The use of autocorrelation analysis in fluorescence correlation spectroscopy (FCS) to monitor hybridization open new degree of sensitivity in the nucleic acid research, however the mass ratio of target strand must be excess about 8 times bigger than labeled strand.Here we try to use dual color correlation method, fluorescence cross correlation spectroscopy, for detection the interaction between same length of double strand and single strand.Materials and MethodCy5- and Rhodamine Green (RG) labeled DNAs of 40 base length, and 20 base were used in this experiment as a model system.Measurements were carried out with the concentration of 20 base, 40 base and control were 2x10-8M, 10-9M and 10-8M, respectively. FCS measurements were carried out wiht ConfoCor 2 (zeiss) with measurement timeof 40 sec.ResultsThe translational diffusion time of both strands (plus and minus) is not sodifferent each other because the length of each strand is equal so that detection of the hybridization product (double strand) by using autocorrelation method (FCS) is very difficult. However, by using the calculation of cross correlation signals from FCCS between both Cy5- and RG, the process of hybridization was detected in a homogeneous solution without any physical separation method. Yield of double strand DNA was calculated about 50% for 40bp and 45% for 20bp with equation. On the other hand, in the case of hybridization between 40 base and 20 base, cross correlation signal was not detected because no complimentary sequence is contained. We now try to detect the formation of triplex DNA in solution by the new FCCS method.