Transcription and reverse transcription of artificial nucleic acids involving backbone modification by template-directed DNA polymerase reactions
Transcription and reverse transcription of artificial nucleic acids involving backbone modification by template-directed DNA polymerase reactions
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DOI：
10.1016/j.bmc.2009.04.045
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发表时间：
2009-06-01
影响因子：
3.5
通讯作者：
Sawai, Hiroaki
中科院分区：
文献类型：
--
作者：
Kuwahara, Masayasu;Takeshima, Hidetoshi;Sawai, Hiroaki
合成了用酰胺型连接物[-CH(2)C@=ONH-]或胺型连接物[-CH(2)CH(2)NH-]取代磷酸二酯连接物的寡脱氧核糖核苷酸（ODN），研究了这些主链改变的影响。聚合酶反应中的阳离子。此外，还合成了一种具有酰胺型连接物的胸苷二核苷酸的三磷酸类似物，并尝试使用这种类似物将酰胺连接物插入到ODN中进行聚合酶反应。引物延伸反应采用三种热稳定性DNA聚合酶KOD（exo-）、Vent（exo-）和Taq进行。对这些数据的分析表明：(1)阳离子的插入对聚合酶反应的影响更大。柔性酰胺型连接剂比插入中性刚性酰胺型连接剂要好；（ii）骨干莫迪。当阳离子靠近引物的3 '端作为延伸端时，它对聚合酶反应的影响比在模板上以及在碱基或糖模中更大。阳离子;（iii）虽然修饰过的DNA模板中的修饰过的连接子被聚合酶越过，但它仍然影响其位置下游几个碱基的延伸反应；（iv）在某些情况下，模板中修饰的连接剂也会影响其位置上游的延伸反应；(v)二核苷酸模拟物掺入需要进一步改进化学结构。2009爱思唯尔有限公司版权所有。
Oligodeoxyribonucleotides (ODN) where the phosphodiester linkage had been replaced with an amide-type linker [-CH(2)C@=ONH-] or an amine-type linker [-CH(2)CH(2)NH-] were synthesized to investigate the effect of these backbone modi. cations on polymerase reactions. In addition, a triphosphate analogue of thymidine dinucleotide with the amide-type linker was synthesized and enzymatic insertion of the amide linkage into ODN was attempted using this analogue for the polymerase reaction. Primer extension reactions using three types of thermostable DNA polymerases, KOD(exo-), Vent(exo-) and Taq were performed for the assays. Analysis of these data indicate that (i) the polymerase reaction tends to be affected much more by insertion of the cationic. exible amine-type linker than by insertion of the neutral rigid amide-type linker; (ii) the backbone modi. cation has a greater effect on the polymerase reaction when it is adjacent to the 3 '-end of a primer as the elongation terminus than when it is on the template, as well as in base or sugar modi. cation; (iii) although the modified linker in the modified DNA template is passed beyond by the polymerase, it still affects the extension reaction several bases downstream from its location; (iv) the modified linker in the template, in some cases, also affects the extension reaction upstream from its location; (v) further improvement of the chemical structure is required for dinucleotide-mimic incorporation. (C) 2009 Elsevier Ltd. All rights reserved.