Polymerase amplification, cloning, and gene expression of benzo-homologous "yDNA" base pairs.
Polymerase amplification, cloning, and gene expression of benzo-homologous "yDNA" base pairs.
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DOI：
10.1002/cbic.200800339
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发表时间：
2008-12-15
期刊：
影响因子：
3.2
通讯作者：
Kool, Eric T.
中科院分区：
文献类型：
--
作者：
Chelliserrykattil, Jijumon;Lu, Haige;Lee, Alex H. F.;Kool, Eric T.
研究了加宽的 DNA 碱基对结构，以探索新的遗传系统设计是否可能拥有天然 DNA 的某些功能的可能性。在“yDNA”系统中，通过添加苯环对配对进行同源，产生（在本研究中）与嘌呤正确配对的苯并嘧啶。在这里，我们报告了苯并嘧啶 yT 和 yC 在体外和细菌细胞中存储和传输生化和生物信息的能力的初步测试。使用两种聚合酶进行的体外引物延伸研究表明，这些酶可以在这些 yDNA 碱基对面插入正确的核苷酸，但选择性较低。使用热稳定性聚合酶进行的 PCR 扩增在 15-20% 的情况下产生了正确的配对，当 yT 或 yC 位于引物内时，更成功。然后将含有一或两个 yDNA 碱基的 DNA 片段连接到质粒中，并测试它们在体内成功引导活性蛋白表达的能力。虽然活性仅为完全天然 DNA 活性的一小部分，但在大多数情况下，当单独取代时，非天然碱基编码正确的密码子碱基，产生功能性绿色荧光蛋白。尽管天然聚合酶对这些大碱基对的活性不大，但这是通过非天然 DNA 碱基对结构在体内编码蛋白质的第一个例子。
A widened DNA base pair architecture is studied in an effort to explore the possibility of whether new genetic system designs might possess some of the functions of natural DNA. In the “yDNA” system, pairs are homologated by addition of a benzene ring, yielding (in the present study), benzopyrimidines that are correctly paired with purines. Here we report initial tests of ability of the benzopyrimidines yT and yC to store and transfer biochemical and biological information in vitro and in bacterial cells. In vitro primer extension studies with two polymerases showed that the enzymes could insert the correct nucleotides opposite these yDNA bases, but with low selectivity. PCR amplifications with a thermostable polymerase resulted in correct pairings in 15–20% of the cases, and more successfully when yT or yC were situated within the primers. Segments of DNA containing one or two yDNA bases were then ligated into a plasmid and tested for their ability to successfully lead the expression of an active protein in vivo. Although active at only a fraction of the activity of fully natural DNA, the unnatural bases encoded the correct codon bases in the majority of cases when singly substituted, yielding functioning green fluorescent protein. Although the activities with native polymerases are modest with these large base pairs, this is the first example of encoding protein in vivo by an unnatural DNA base pair architecture.
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DOI：
10.1073/pnas.92.14.6329
发表时间：
1995-07-03
影响因子：
11.1
作者：
HORLACHER, J;HOTTIGER, M;BENNER, SA
通讯作者：
BENNER, SA
影响因子：
64.8
作者：
BAIN, JD;SWITZER, C;BENNER, SA
通讯作者：
BENNER, SA
影响因子：
4.3
作者：
Lai, JS;Kool, ET
通讯作者：
Kool, ET
影响因子：
14.9
作者：
Johnson, SC;Sherrill, CB;Prudent, JR
通讯作者：
Prudent, JR
影响因子：
4.8
作者：
Kim, TW;Brieba, LG;Kool, ET
通讯作者：
Kool, ET