Nonenzymatic Ligation of DNA with a Reversible Step and a Final Linkage that Can Be Used in PCR
Nonenzymatic Ligation of DNA with a Reversible Step and a Final Linkage that Can Be Used in PCR
复制标题
DOI：
10.1002/cbic.201200167
复制
发表时间：
2012-05-29
期刊：
影响因子：
3.2
通讯作者：
Hud, Nicholas V.
中科院分区：
文献类型：
--
作者：
Engelhart, Aaron E.;Cafferty, Brian J.;Hud, Nicholas V.
DNA现在被用于远远超出其自然信息承载能力的目的（例如，在纳米技术中，作为催化剂和识别元件）。[1]因此，这种聚合物出现在传感器、治疗剂和成像技术等各种应用中。[1e用于与水溶液中的自组装相容的寡核苷酸的非酶促偶联的合成方法，允许选择生物学上有利的组装，进一步扩展了DNA的可能用途。[3]然而，用于核酸的非酶促聚合的大多数方法缺乏允许热力学选择的手段，涉及大多数实验室无法获得的合成程序，或导致形成聚合酶无法读取的聚合物键，从而限制了现有连接方法的可及性和实用性。在此，我们报道了一种DNA连接系统，该系统：1）使用市售试剂可获得; 2）包括可允许选择生物学上有利的产物的可逆步骤; 3）在较低浓度下以模板选择性模式进行; 4）在较高底物浓度下以非模板模式进行;和5）通过许多嗜热和嗜温聚合酶在模板链中产生可耐受的连接，允许通过PCR扩增产物序列。我们已经研究了DNA双链体和DNA聚合酶对吗啉骨架键合（1）的相容性，吗啉骨架键合（1）可以通过含有5 ′-氨基残基的寡核苷酸与含有高碘酸氧化的3 ′-核糖的寡核苷酸反应形成（方案1）。一段时间以来，已知通过核糖核苷酸的高碘酸盐氧化产生的2 ′，3 ′-二醛在水中与烷基胺反应，在还原时产生水解稳定的吗啉。[4]Wincott及其同事先前已经证明了这种胺-二醛连接反应在RNA发夹环中的实用性，[5]其他研究人员已经使用类似的反应将化学基团延伸和缀合到核酸的末端。[5-6]然而，据我们所知，吗啉键对DNA双链体稳定性和聚合酶模板链的影响尚未报道。这里研究的连接与众所周知的磷酰二胺吗啉代连接[7]有很大的不同，因为连接1在吗啉环和其3 '侧的残基之间缺少磷酰二胺基团（方案1）。探索连锁1和相关的未还原连锁2的可能性（方案1）为了支持在天然连接的DNA内形成双链体，将五聚体d（GAGT）rC（Ald 5）与NaIO 4在5 ′-氨基-d（TAAGC）（Am 5）和十聚体d的存在下，（GCTTAGACTC）（Temp 10），其可以用作连接Ald 5和Am 5的模板。选择这些寡核苷酸长度，使得大多数Ald 5和Am 5在孵育条件下与Temp 10杂交（每条链中1 mM，58 C），[8]如果连接1或2与DNA双链体相容，则杂交复合物进一步稳定。连接2的稳定作用通过以下事实证明：在不存在还原剂Ald 5的情况下，Am 5和Temp 10形成复合物，当在分析前立即稀释500倍时，其表现出协同解链转变（Tm）！208 C（参见支持信息中的图S1-
DNA is now used for purposes well beyond its natural information-carrying capacity (eg, in nanotechnology, as a catalyst, and as a recognition element).[1] As a result, this polymer appears in applications as varied as sensors, therapeutic agents, and imaging technologies.[1e, 2] Synthetic procedures for the nonenzymatic coupling of oligonucleotides that are compatible with self-assembly in aqueous solution, allowing selection of thermodynamically favored assemblies, have further expanded the possible uses of DNA.[3] However, most methods used for the nonenzymatic polymerization of nucleic acids lack a means to allow thermodynamic selection, involve synthetic procedures that are inaccessible to most laboratories, or result in formation of a polymer linkage that polymerase enzymes cannot read, thereby limiting the accessibility and utility of existing ligation methods. Here, we report a DNA ligation system that: 1) Is accessible using commercially available reagents; 2) includes a reversible step that can allow selection of a thermodynamically favored product; 3) proceeds in a templateselective mode at lower concentrations; 4) proceeds in an untemplated mode at higher substrate concentrations; and 5) produces a linkage that can be tolerated in a template strand by a number of thermophilic and mesophilic polymerases, allowing product sequence amplification by PCR. We have investigated the DNA duplex and DNA polymerase compatibilities of the morpholine backbone linkage (1) that can be formed by the reaction of an oligonucleotide containing a 5’-amino residue with an oligonucleotide containing a periodate-oxidized 3’-ribose (Scheme 1). It has been known for some time that the 2’, 3’-dialdehydes generated by periodate oxidation of ribonucleotides react in water with alkylamines to produce, upon reduction, a hydrolytically stable morpholine.[4] Wincott and co-workers have previously shown the utility of this amine-dialdehyde ligation reaction within an RNA hairpin loop,[5] and other investigators have used similar reactions to extend and conjugate chemical groups onto the ends of nucleic acids.[5–6] However, to the best of our knowledge, the effect of a morpholine linkage on DNA duplex stability and in a polymerase template strand has not been reported. The linkage investigated here differs substantially from the well-known phosphorodiamidate morpholino linkage [7] in that linkage 1 lacks a phosphorodiamidate group between the morpholine ring and the residue on its 3’side (Scheme 1). To explore the potential for linkage 1 and the related unreduced linkage 2 (Scheme 1) to support duplex formation within otherwise naturally linked DNA, the pentamer d (GAGT) rC (Ald5) was incubated with NaIO4 (to generate the 2’, 3’-dialdehyde) in the presence of 5’-amino-d (TAAGC)(Am5) and the decamer d (GCTTAGACTC)(Temp10), which could serve as a template for the ligation of Ald5 and Am5. These oligonucleotide lengths were chosen such that the majority of Ald5 and Am5 would hybridize with Temp10 under the conditions of incubation (1 mM in each strand, 58C),[8] with the hybridized complex being further stabilized if either linkage 1 or 2 is compatible with a DNA duplex.The stabilizing effect of linkage 2 is demonstrated by the fact that, in the absence of a reducing agent, Ald5, Am5, and Temp10 form a complex, which, when diluted 500-fold immediately before analysis, exhibits a cooperative melting transition (Tm) of! 208C (see Figure S1 in the Supporting Informa-