Sequence-specific scission of DNA by the chemical nuclease activity of 1,10-phenanthroline-copper(I) targeted by RNA.
Sequence-specific scission of DNA by the chemical nuclease activity of 1,10-phenanthroline-copper(I) targeted by RNA.
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RNA 靶向的 1,10-菲咯啉-铜 (I) 的化学核酸酶活性对 DNA 进行序列特异性切割。
DOI：
10.1073/pnas.90.9.4206
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发表时间：
1993
影响因子：
11.1
通讯作者：
Sigman,DS
中科院分区：
文献类型：
--
作者：
Chen,CB;Gorin,MB;Sigman,DS
用化学核酸酶1,10-菲罗啉-铜修饰的rna可以实现单链和双链DNA靶标的序列特异性切割。该rna以5-（3-氨基丙烯基）-UTP作为UTP的唯一来源在体外制备，并可通过n -琥珀酰亚胺基3-（2-吡啶基二硫代）丙酸酯（SPDP）与1,10-邻菲罗啉进行修饰，将配体交联至氨基丙烯基部分。单链dna在多个位点被有效地切割，因为1,10-菲罗啉在序列中的几个尿嘧啶上被结合。序列特异性的双链DNA切割也可以在R环内由1,10-邻菲罗啉衍生的RNA完成。这些三链结构在70%的甲酰胺中形成，并涉及由相同序列的RNA取代一条DNA链。R环定向切割是DNA切割的第一种方法，适用于任何序列。R环靶向核裂变的一个独特应用是确定靶标内两个标记DNA序列之间的距离，它依赖于它在任何序列上切割DNA的能力。在这种情况下，1,10-菲罗啉连接的rna由两个不同的序列制备，并用于在r环形成后切割DNA片段。通过这些方法释放的片段的大小是两个DNA序列之间距离的碱基对的直接测量。例如，用这种方法确定了两个鸡δ结晶蛋白（δ 1和δ 2）基因之间的距离为24千碱基。
RNAs modified with the chemical nuclease 1,10-phenanthroline-copper(I) can achieve the sequence-specific scission of single- and double-stranded DNA targets. The RNAs are prepared in vitro by using 5-(3-aminoallyl)-UTP as the sole source of UTP and can be readily modified with 1,10-phenanthroline by using N-succinimidyl 3-(2-pyridyl-dithio)propionate (SPDP) to cross-link the ligand to the aminoallyl moiety. Single-stranded DNAs are efficiently cleaved at multiple sites because 1,10-phenanthroline is incorporated at several uridines in the sequence. Sequence-specific double-stranded scission of duplex DNA can also be accomplished with 1,10-phenanthroline-derivatized RNA within R loops. These triple-stranded structures form in 70% formamide and involve the displacement of one strand of DNA by the RNA of identical sequence. R loop-directed scission is the first method for DNA scission applicable to any sequence. A unique application of R loop-targeted nucleolytic scission, which relies on its ability to cut DNA at any sequence, is the determination of the distance between two marker DNA sequences within a target. In this case, 1,10-phenanthroline-linked RNAs are prepared from the two distinct sequences and used to cut the DNA fragment after R-loop formation. The size of the fragment liberated by these methods is a direct measure in base pairs of the distance between the two DNA sequences. For example, the distance separating two chicken delta crystallin (delta 1 and delta 2) genes has been confirmed as 24 kilobases by this method.