Misra H S, Pandey P K, Modak M J, Vinayak R, Pandey V N
Department of Biochemistry and Molecular Biology, UMD-New Jersey Medical School, Newark 07103, USA.
Biochemistry. 1998 Feb 17;37(7):1917-25. doi: 10.1021/bi971524n.
A peptide nucleic acid (PNA) oligomer, an analogue of DNA, was examined for its ability to function as a primer or a template to support DNA synthesis catalyzed by DNA polymerases. The analogue, (PNA)19-TpG-OH, comprised of 19 bases in the form of PNA followed by a dinucleotide (TpG-OH) with a single phosphate and a free 3'OH terminus, was recognized as a bona fide primer by 2 reverse transcriptases and also by the Klenow fragment of E. coli DNA polymerase I. The 21-mer PNA chimera is extended on both RNA and DNA templates by all three polymerases. The specificity of binding of the PNA chimeric primer/DNA template at the template-primer binding site of the enzyme was shown by its photo-cross-linking ability to the enzyme which could be effectively competed out by another TP but not by template or primer alone. Furthermore, the chimeric TP-enzyme covalent complex was found to be catalytically active as judged by its ability to incorporate one nucleotide onto the 3'OH terminus of the immobilized primer. PNA sequences were also recognized as template when annealed with a DNA primer. These observations are in variance with the conventional suggestion that the phosphate backbone in the duplex region is essential for recognition and binding by DNA polymerases. The efficient extension of (PNA)19-TpG-OH suggests that the diameter of the duplex region of template primer rather than the phosphate backbone may be sufficient for recognition by DNA polymerases.
对一种肽核酸（PNA）寡聚物（DNA的类似物）作为引物或模板支持DNA聚合酶催化DNA合成的能力进行了研究。该类似物（PNA）19-TpG-OH由19个PNA形式的碱基组成，后面接一个带有单磷酸和游离3'OH末端的二核苷酸（TpG-OH），被两种逆转录酶以及大肠杆菌DNA聚合酶I的Klenow片段识别为真正的引物。这种21聚体PNA嵌合体在RNA和DNA模板上均能被所有三种聚合酶延伸。PNA嵌合引物/DNA模板在酶的模板-引物结合位点的结合特异性通过其与酶的光交联能力得以体现，该能力可被另一种模板引物有效竞争，但不能被单独的模板或引物竞争。此外，通过将一个核苷酸掺入固定化引物的3'OH末端的能力判断，发现嵌合模板引物-酶共价复合物具有催化活性。当与DNA引物退火时，PNA序列也被识别为模板。这些观察结果与传统观点不同，传统观点认为双链区域中的磷酸骨架对于DNA聚合酶的识别和结合至关重要。（PNA）19-TpG-OH的有效延伸表明，模板引物双链区域的直径而非磷酸骨架可能足以被DNA聚合酶识别。
