High-purity preparation of a large DNA dumbbell
High-purity preparation of a large DNA dumbbell
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DOI：
10.1089/108729001300338672
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发表时间：
2001-06-01
期刊：
影响因子：
--
通讯作者：
Demidov, VV
中科院分区：
文献类型：
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作者：
Kuhn, H;Frank-Kamenetskii, MD;Demidov, VV
我们报告了一个大的DNA哑铃从一对短的DNA发夹与长的单链尾巴的任意序列的有效的生化合成。通过酶促连接获得DNA哑铃，产生在两个末端由5 nt的单链环封闭的94-bp双链体茎。连接后，所有未连接的前体和单连接的副产物通过切口平移或引物延伸或两者被多重生物素化。因此，它们可以容易地通过温和的生物磁性分离程序从DNA哑铃状制备物中除去。纯化的DNA哑铃的封闭构象通过其改变的凝胶迁移率与未连接或monoligated样品相比，并通过核酸外切酶测定来验证。考虑到DNA哑铃有前途的治疗潜力，开发的生物合成方法可用于高纯度制备更长的共价闭合的DNA诱饵。
We report on the efficient biochemical synthesis of a large DNA dumbbell starting from a pair of short DNA hairpins with long single-stranded tails of arbitrary sequence. The DNA dumbbell is obtained by enzymatic ligation yielding a 94-bp duplex stem closed at both termini by single-stranded loops of 5 nt, Following ligation, all unligated precursors and monoligated by-products were multiply biotinylated via nick-translation or primer-extension or both. Thus, they could readily be removed from the DNA dumbbell preparation by a mild biomagnetic separation procedure. The closed conformation of the purified DNA dumbbell was verified by its altered gel mobility as compared with unligated or monoligated samples and by an exonuclease assay. Considering the promising therapeutic potential of DNA dumbbells, the developed biosynthetic approach could be used for high-purity preparation of longer, covalently closed DNA decoys.