Landgraf R, Chen C H, Sigman D S
Department of Biological Chemistry, School of Medicine, University of California-Los Angeles 90024-1570, USA.
Nucleic Acids Res. 1995 Sep 11;23(17):3516-23. doi: 10.1093/nar/23.17.3516.
The sequence-specific formation of R-loops can be assayed using RNAs which overlap a HindIII cleavage site in a 3.5 kb plasmid. Chemical modification of the displaced DNA strand has permitted stabilization of these R-loops and allowed a systematic investigation of the dependence of these triple-stranded structures on the chain length and structure of the input RNA. RNAs as short as 50 nt form stable R-loops if 5-allylamine uridines (Uaa-RNA) are used in place of normal uridines; normal RNAs must be 100 nt long to form R-loops quantitatively. Since acetic anhydride decreases the hybridization efficiency of Uaa-RNAs, the positive charge of the RNAs must diminish the electrostatic repulsion of the three negatively charged phosphodiester backbones. The dependence of R-loop stability on the length of RNA can be stimulated with a random walk model, which also applies to strand migration within Holiday junctions. R-loop hybridization provides a versatile method to generate single-stranded DNA in a sequence-selective manner.
R环的序列特异性形成可以使用与3.5 kb质粒中HindIII切割位点重叠的RNA进行检测。对置换的DNA链进行化学修饰可以使这些R环稳定，并允许对这些三链结构对输入RNA的链长和结构的依赖性进行系统研究。如果使用5-烯丙基胺尿苷（Uaa-RNA）代替正常尿苷，短至50 nt的RNA也能形成稳定的R环；正常RNA必须100 nt长才能定量形成R环。由于乙酸酐会降低Uaa-RNA的杂交效率，RNA的正电荷必定会减少三条带负电荷的磷酸二酯主链之间的静电排斥。R环稳定性对RNA长度的依赖性可以用随机游走模型来模拟，该模型也适用于霍利迪连接体中的链迁移。R环杂交提供了一种以序列选择性方式生成单链DNA的通用方法。
