Iyer M, Norton J C, Corey D R
Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas 75235, USA.
J Biol Chem. 1995 Jun 16;270(24):14712-7. doi: 10.1074/jbc.270.24.14712.
We report two strategies for accelerating the hybridization of oligonucleotides to DNA. We demonstrate that oligodeoxyribonucleotides and peptide nucleic acid oligomers hybridize to inverted repeats within duplex DNA by D-loop formation. Oligonucleotides and duplex template form an active complex, which can be recognized by T7 DNA polymerase to prime polymerization. Quantitation of polymerization products allowed the rate of hybridization to be estimated, and peptide nucleic acid oligomers and oligonucleotide-protein adducts anneal with association constants 500- and 12,000-fold greater, respectively, than the analogous unmodified oligonucleotides. Together, these results indicate that sequences within duplex DNA can be targeted by Watson-Crick base pairing and that chemical modifications can dramatically enhance the rate of strand association. These findings should facilitate targeting of oligomers for priming DNA polymerization, the detection of diagnostic sequences, and the disruption of gene expression. The observed acceleration of hybridization may offer a new perspective on the ability of RecA or other proteins to accelerate strand invasion.
我们报告了两种加速寡核苷酸与DNA杂交的策略。我们证明了寡脱氧核糖核苷酸和肽核酸寡聚物通过D环形成与双链DNA内的反向重复序列杂交。寡核苷酸和双链模板形成一个活性复合物，可被T7 DNA聚合酶识别以引发聚合反应。对聚合产物的定量分析使得杂交速率得以估算，并且肽核酸寡聚物和寡核苷酸-蛋白质加合物的退火结合常数分别比类似的未修饰寡核苷酸大500倍和12000倍。这些结果共同表明，双链DNA内的序列可通过沃森-克里克碱基配对来靶向，并且化学修饰可显著提高链结合速率。这些发现应有助于将寡聚物靶向用于引发DNA聚合反应、检测诊断序列以及破坏基因表达。观察到的杂交加速可能为RecA或其他蛋白质加速链侵入的能力提供新的视角。
