Single-molecule mechanics of protein-labelled DNA handles.
Single-molecule mechanics of protein-labelled DNA handles.
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DOI：
10.3762/bjnano.7.16
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发表时间：
2016
影响因子：
3.1
通讯作者：
Hegner M
中科院分区：
文献类型：
--
作者：
Jadhav VS;Brüggemann D;Wruck F;Hegner M
在单分子实验中，DNA手柄通常被用作间隔物和连接物，用于分离和锚定表面修饰的珠子之间的RNA、蛋白质、酶和核酶等生物分子，以进行纳米机械研究。具有不同长度和化学末端修饰的定制DNA手柄可以轻松可靠地整体合成，从而能够在大范围的外力作用下，在生理条件下进行定义明确和持久的力谱测量。尽管这些化学标记的DNA手柄被广泛使用，但它们进一步用蛋白质受体进行个体修饰的情况并不常见，并且可以在抓取生物分子进行机械测量时提供额外的灵活性。关于这些DNA-蛋白质杂交体合成的可靠方案以及它们在不同生理条件下的机械特性的深入信息在文献中是缺乏的。在这里，我们使用光学镊子在不同的生理条件下，在微流控环境中研究不同蛋白质标记的DNA手柄。将不同大小(1000、3034和4056bp)的地高辛(Dig)-双链DNA-生物素手柄与链霉亲和素或中性亲和素蛋白偶联。这些杂化材料的DIG修饰末端结合到表面修饰的聚苯乙烯(反DIG)小球上。使用不同的生理缓冲液，光学力测量显示出一致的力学特性和较长的解离时间。这些蛋白质修饰的DNA杂交物也与其他生物素化的DNA分子在原位相互连接。电子倍增电荷耦合器件(EMCCD)成像控制实验表明，DNA手柄末端的量子点-链霉亲和素偶联物仍然可以自由访问。这里介绍的实验证明，用我们的蛋白质-DNA标记程序生产的手柄是在时间关键的分子马达研究中抓住暴露适合分子识别的标签的单分子的极佳候选者。
DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time-critical molecular motor studies.