Triazine-Modified 7-Deaza-2'-deoxyadenosines: Better Suited for Bioorthogonal Labeling of DNA by PCR than 2'-Deoxyuridines.
Triazine-Modified 7-Deaza-2'-deoxyadenosines: Better Suited for Bioorthogonal Labeling of DNA by PCR than 2'-Deoxyuridines.
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DOI：
10.1021/acs.bioconjchem.9b00295
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发表时间：
2019-05
影响因子：
4.7
通讯作者：
Ulrike Reisacher;Bastian Groitl;Ralf Strasser;G. Cserép;P. Kele;H. Wagenknecht
中科院分区：
文献类型：
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作者：
Ulrike Reisacher;Bastian Groitl;Ralf Strasser;G. Cserép;P. Kele;H. Wagenknecht
6-乙炔基-1，2，4-三嗪是一种具有生物正交反应活性的小基团，我们将其应用于具有反电子需求的Diels-Alder反应中寡核苷酸的荧光标记。我们通过合成将该官能团连接到7-脱氮-2 '-脱氧腺苷三磷酸的7-位和2'-脱氧尿苷三磷酸的5-位。两种修饰的核苷酸三磷酸被用于引物延伸实验（PEX）和PCR扩增的比较，最终产生多标记的寡核苷酸的合成后反应与高反应性的双环[6.1.0]壬炔-罗丹明共轭物。这些实验表明，与连接在2 ′-脱氧尿苷的5-位上相比，当连接在7-脱氮-2 ′-脱氧腺苷的7-位上时，DNA聚合酶对6-乙炔基-1，2，4-三嗪的耐受性好得多。这通过PCR产物的PAGE分析和使用switchSENSE在引物延伸期间DNA聚合酶活性的实时动力学观察变得明显。一般来说，我们的研究结果表明，生物正交标记策略更适合7-脱氮-2 '-腺苷比传统的和可用的2'-脱氧尿苷。
6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels-Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate and to the 5-position of 2'-deoxyuridine triphosphate. Both modified nucleotide triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA polymerase when attached to the 7-position of 7-deaza-2'-deoxyadenosine in comparison to the attachment at the 5-position of 2'-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.