Getting swept off your toe(hold)s: Single-molecule DNA fission analysis offers glimpse into kinetics of branch migration.
Getting swept off your toe(hold)s: Single-molecule DNA fission analysis offers glimpse into kinetics of branch migration.
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DOI：
10.1016/j.bpj.2021.04.014
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发表时间：
2021-04
影响因子：
3.4
通讯作者：
M. Spies
中科院分区：
文献类型：
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作者：
M. Spies
碱基配对的可预测性、直接性和双螺旋的物理性质易于理解，使得核酸（DNA 和 RNA）成为组装复杂纳米结构以及非平衡纳米器件的通用工具。通过改变构建块的顺序，人们几乎可以组装任何可以想象到的几何形状和图案。在生理盐浓度和温度下，B 型 DNA 和 A 型 RNA 的双螺旋本质上是热力学稳定的。然而，当部分双链体与与双链体中较长链互补的入侵单链混合时，可以发生链交换，从而允许构建活性装置，其中核酸既用作结构材料又用作燃料（1）。这种涉及互补或部分互补序列的链置换反应被称为“立足点介导的”链置换，因为该反应是通过将入侵链与底物链上的短单链突出区域（“立足点”）配对而引发的。立足点配对之后，将现有的互补链逐出至底物，称为“现有”链（图 1）。链位移是许多纳米技术应用的基石，包括复杂纳米结构的自组装、纳米级电路、自主步行器、可调谐纳米器件、基因突变和多态性的诊断以及合成生物学应用中的可编程开关（请参阅（3）了解最近的评论）。在自然界中，链置换和分支迁移通常是许多 DNA 和 RNA 代谢过程的共同特征，例如同源基因重组过程中 D 环的形成、断裂 DNA 复制叉的重排、霍利迪连接体的分支迁移、RNA 转录中 R 环的形成和加工以及 CRISPR-Cas 系统的序列识别等。在立足点介导的链置换反应中，立足点的形成是限速步骤。这也是最好理解的步骤，因为可以通过改变立足点长度（通常在 2 到 8 个核苷酸之间）、立足点序列和缓冲条件来轻松控制反应速率。增加立足点长度通常可以通过增加缔合速率来加速反应，并且还可以通过使产物比底物在热力学上更有利来推动反应前进。人们还可以通过故意“隐藏”立足点结构域来控制立足点介导的链位移。这可以通过将悬挂的立足点与可移除的寡核苷酸杂交，将其定位在发夹或三链体形成结构内，或通过核碱基笼蔽（在（3）中综述）来实现。总反应速率显示出对立足点长度的指数依赖性，表明初始配对是限速步骤 (4)，并且总体立足点介导的链置换反应通常被建模为双分子关联。此外，三步模型已用于根据 DNA 杂交的热力学定量预测链置换动力学 (5)。该模型考虑了两个参数，即 DNA 杂交和分支迁移的速率常数，并且对于没有形成二级结构潜力的序列相当有效 (5)。所提出的立足点介导的链置换的能量景观假设启动分支迁移会受到轻微的惩罚（25°C 时为 2 kcal/mol）(6)，之后分支迁移以随机的单碱基对步骤进行……
The predictable, straightforward nature of basepairing and the well-understood physical properties of the double helix make nucleic acids (DNA and RNA) versatile tools for assembly of complex nanostructures as well as nonequilibrium nanodevices. By varying sequences of building blocks, one can assemble virtually any imaginable geometric shape and pattern. At physiological salt concentrations and temperatures, double helixes of B-form DNA and A-form RNA are inherently thermodynamically stable. When, however, a partial duplex is mixed with an invading single strand complementary to the longer strand in the duplex, an exchange of the strands can take place, thus allowing construction of active devices where nucleic acids are used as both structural materials and as a fuel (1). Such strand displacement reactions involving complementary or partially complementary sequences are referred to as ‘‘toeholdmediated’’strand displacement, as the reaction is initiated by pairing of the invading strand to a short singlestranded overhang region(a ‘‘toehold’’) on the substrate strand. The toehold pairing is then followed by eviction of the existing complement to the substrate, known as the ‘‘incumbent’’strand (Fig. 1). Strand displacement is a cornerstone of many nanotechnology applications including self-assembly of complex nanostructures, nanoscale circuits, autonomous walkers, tunable nanodevices, diagnostics for the presence of genetic mutations and polymorphisms, and programmable switches in synthetic biology applications (see (3) for a recent review). In nature, strand displacement, and branch migration in general, is a feature common to many processes of DNA and RNA metabolism such as formation of D-loops during homologous genetic recombination, rearrangements of broken DNA replication forks, branch migration of Holliday junctions, formation and processing of R-loops in RNA transcription, and sequence recognition by the CRISPR-Cas system, among many others. In toehold-mediated strand displacement reactions, the toehold formation is a rate-limiting step. It is also the best-understood step, as one can easily control the reaction rate by changing the toehold length (usually between two and eight nucleotides), toehold sequence, and buffer conditions. Increasing the toehold length generally speeds up the reaction by increasing the association rate and also drives the reaction forward by making the products more thermodynamically favorable than the substrates. One can also control the toehold-mediated strand displacement by deliberately ‘‘hiding’’the toehold domain. This can be achieved by hybridization of the dangling toehold with a removable oligonucleotide, positioning it within a hairpin or triplex forming structure, or by nucleobase caging (reviewed in (3)). The overall reaction rate displays an exponential dependence on the toehold length suggesting that the initial pairing is the rate-limiting step (4), and the overall toehold-mediated strand displacement reaction is commonly modeled as a bimolecular association. Further, a three-step model has been used to quantitatively predict strand displacement kinetics from thermodynamics of DNA hybridization (5). This model takes into account two parameters, which are the rate constants for DNA hybridization and for branch migration, and works reasonably well for sequences devoid of the potential to form secondary structures (5). The proposed energy landscapes for the toehold-mediated strand displacement assume that there is a slight penalty to initiate branch migration (2 kcal/mol at 25 C)(6), after which branch migration proceeds in single basepair steps by a random one …