Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation
Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation
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减少核酸模板化连接反应中的产物抑制：DNA模板化环化
DOI：
10.1002/cbic.201300516
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发表时间：
2013
期刊：
影响因子：
3.2
通讯作者：
O. Seitz
中科院分区：
文献类型：
--
作者：
A. Roloff;O. Seitz
可编程相互作用允许核酸分子通过增加附加反应基团的有效摩尔数来模板化化学反应。原则上，DNA/RNA引发的反应可以通过模板的翻转来进行。当DNA或RNA靶标的可用性有限时，每个模板形成许多产物分子所提供的扩增是一项宝贵的资产。然而，由于反应产物阻碍了模板的使用，通常会阻碍其周转。产物抑制在连接反应中最严重，连接后的产物极大地增加了模板亲和力。我们介绍了一种潜在的通用方法来减少核酸程序连接反应中的产物抑制。DNA触发的双功能肽核酸(PNA)结合物的连接-环化序列(“环化”)提供了环化连接产物。熔融实验表明，与线性连接产物相比，产物环化伴随着模板亲和力的显著降低。该反应体系依赖于卤代乙酰化的PNA-硫代酯和异半胱氨酸基-PNA-半胱氨酸结合物，它们按照天然的化学连接机制连接到DNA模板上。所得到的线性产物-模板双链体的解离(例如由热循环引起)使产物能够通过硫卤化物取代而环化。连接反应和环合反应都是快速反应(连接反应：20分钟后产率为86 %，环合反应：5分钟后为定量反应)。在热循环条件下，当加入新的反应物时，DNA模板能够触发新产物分子的形成。此外，环化反应产生的产物是使用亚化学计量比模板负载(0.25-0.01当量)的传统连接反应的2-3倍。我们相信，DNA模板反应产物的环化最终可能提供完全克服产物抑制的系统。
Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups. DNA/RNA‐triggered reactions can proceed, in principle, with turnover in the template. The amplification provided by the formation of many product molecules per template is a valuable asset when the availability of the DNA or RNA target is limited. However, turnover is usually impeded by reaction products that block access to the template. Product inhibition is most severe in ligation reactions, where products after ligation have dramatically increased template affinities. We introduce a potentially generic approach to reduce product inhibition in nucleic acid‐programmed ligation reactions. A DNA‐triggered ligation–cyclization sequence (“cycligation”) of bifunctional peptide nucleic acid (PNA) conjugates affords cyclic ligation products. Melting experiments revealed that product cyclization is accompanied by a pronounced decrease in template affinity compared to linear ligation products. The reaction system relies upon haloacetylated PNA‐thioesters and isocysteinyl‐PNA‐cysteine conjugates, which were ligated on a DNA template according to a native chemical ligation mechanism. Dissociation of the resulting linear product‐template duplex (induced by, for example, thermal cycling) enabled product cyclization through sulfur‐halide substitution. Both ligation and cyclization are fast reactions (ligation: 86 % yield after 20 min, cyclization: quantitative after 5 min). Under thermocycling conditions, the DNA template was able to trigger the formation of new product molecules when fresh reactants were added. Furthermore, cycligation produced 2–3 times more product than a conventional ligation reaction with substoichiometric template loads (0.25–0.01 equiv). We believe that cyclization of products from DNA‐templated reactions could ultimately afford systems that completely overcome product inhibition.
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影响因子：
56.9
作者：
A. Schwartz;L. Orgel
通讯作者：
L. Orgel
模板指导的 PNA 连接反应的序列保真度。
影响因子：
4.9
作者：
Amos Mattes;O. Seitz
通讯作者：
O. Seitz
影响因子：
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作者：
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通讯作者：
G. V. Kiedrowski
影响因子：
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作者：
Kleiner RE;Dumelin CE;Liu DR
通讯作者：
Liu DR
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影响因子：
1.8
作者：
Xu,Yanzheng;Kool,EricT
通讯作者：
Kool,EricT