Selective nucleic acid capture with shielded covalent probes.
Selective nucleic acid capture with shielded covalent probes.
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DOI：
10.1021/ja4009216
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发表时间：
2013-07-03
影响因子：
15
通讯作者：
Pierce, Niles A.
中科院分区：
文献类型：
--
作者：
Vieregg, Jeffrey R.;Nelson, Hosea M.;Stoltz, Brian M.;Pierce, Niles A.
核酸探针用于体外、原位和体内的各种应用。在任何情况下，它们的能力都受到不完美选择性（结合不需要的目标）和不完全亲和性（结合是可逆的，并不是所有想要的目标都结合）的限制。这些困难是根本性的，源于依赖碱基配对来提供选择性和亲和力。屏蔽共价（SC）探针消除了选择性和持久目标捕获之间的长期权衡，通过可编程碱基配对和分子构象改变实现选择性，并通过可激活的共价交联实现持久目标捕获。在纯样品和混合样品中，SC探针共价捕获互补的DNA或RNA寡核苷酸靶标，并在室温下以接近定量的产量拒绝双核苷酸不匹配的靶标，实现2-3个数量级的鉴别比。全长mRNA靶点的半定量研究表明，选择性共价捕获与RNA寡核苷酸靶点相当。单核苷酸DNA或RNA错配，包括几乎等能的RNA摆动对，可以有效地以1-2个数量级的辨别率被拒绝。共价捕获率符合探针/目标杂化的热力学规律，有利于合理设计探针。如果需要，可以反转交联以在捕获后释放目标。与现有的探针化学相比，SC探针实现了结构化探针的高序列选择性，但即使在变性条件下也能持久地保留其目标。这种先前不相容的性质组合表明，在碱基配对被破坏的条件下（例如，通过体外或原位严格洗涤，或通过体内酶），基于核酸靶点的选择性和稳定结合的多种应用。
Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible, and not all desired targets bound). These difficulties are fundamental, stemming from reliance on base pairing to provide both selectivity and affinity. Shielded covalent (SC) probes eliminate the longstanding trade-off between selectivity and durable target capture, achieving selectivity via programmable base pairing and molecular conformation change, and durable target capture via activatable covalent cross-linking. In pure and mixed samples, SC probes covalently capture complementary DNA or RNA oligo targets and reject two-nucleotide mismatched targets with near-quantitative yields at room temperature, achieving discrimination ratios of 2–3 orders of magnitude. Semiquantitative studies with full-length mRNA targets demonstrate selective covalent capture comparable to that for RNA oligo targets. Single-nucleotide DNA or RNA mismatches, including nearly isoenergetic RNA wobble pairs, can be efficiently rejected with discrimination ratios of 1–2 orders of magnitude. Covalent capture yields appear consistent with the thermodynamics of probe/target hybridization, facilitating rational probe design. If desired, cross-links can be reversed to release the target after capture. In contrast to existing probe chemistries, SC probes achieve the high sequence selectivity of a structured probe, yet durably retain their targets even under denaturing conditions. This previously incompatible combination of properties suggests diverse applications based on selective and stable binding of nucleic acid targets under conditions where base-pairing is disrupted (e.g., by stringent washes in vitro or in situ, or by enzymes in vivo).
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DOI：
10.1073/pnas.2233244100
发表时间：
2003-11-11
影响因子：
11.1
作者：
Bratu, DP;Cha, BJ;Tyagi, S
通讯作者：
Tyagi, S
影响因子：
64.8
作者：
Castanotto, Daniela;Rossi, John J.
通讯作者：
Rossi, John J.
影响因子：
14.9
作者：
Li, QQ;Luan, GY;Liang, JX
通讯作者：
Liang, JX
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
5.6
作者：
Biswas, I;Yamamoto, A;Hsieh, P
通讯作者：
Hsieh, P