Department of Chemical and Biological Engineering, Polytechnic Institute of New York University, 6 MetroTech Center, Brooklyn, New York 11201, USA.
J Am Chem Soc. 2011 Aug 3;133(30):11588-96. doi: 10.1021/ja202631b. Epub 2011 Jul 12.
Morpholinos (MOs) are DNA analogues whose uncharged nature can bring fundamental advantages to surface hybridization technologies such as DNA microarrays, by using MOs as the immobilized, or "probe", species. Advancement of MO-based diagnostics, however, is challenged by limited understanding of the surface organization of MO molecules and of how this organization impacts hybridization kinetics and thermodynamics. The present study focuses on hybridization kinetics between monolayers of MO probes and DNA targets as a function of the instantaneous extent of hybridization (i.e., duplex coverage), total probe coverage, and ionic strength. Intriguingly, these experiments reveal distinct kinetic stages, none of which are consistent with Langmuir kinetics. The initial stage, in which duplex coverage remains relatively sparse, indicates confluence of two effects: blockage of target access to unhybridized probes by previously formed duplexes and deactivation of the solid support due to consumption of probe molecules. This interpretation is consistent with a surface organization in which unhybridized MO probes localize near the solid support, underneath a layer of MO-DNA duplexes. As duplex coverage builds, provided saturation is not reached first, the initial stage can transition to an unusual regime characterized by near independence of hybridization rate on duplex coverage, followed by a prolonged approach to equilibrium. The possible origins of these more complex latter behaviors are discussed. Comparison with published data for DNA and peptide nucleic acid (PNA) probes is carried out to look for universal trends in kinetics. This comparison reveals qualitative similarities when comparable surface organization of probes is expected. In addition, MO monolayers are found capable of a broad range of reactivities that span reported values for PNA and DNA probes.
吗啉代寡核苷酸（MOs）是不带电荷的 DNA 类似物，这一特性使其在 DNA 微阵列等表面杂交技术中具有基本优势，因为可以将 MO 用作固定的“探针”物质。然而，基于 MO 的诊断技术的发展受到 MO 分子表面结构的理解有限以及这种结构如何影响杂交动力学和热力学的限制。本研究主要关注 MO 探针单层与 DNA 靶标的杂交动力学，其影响因素包括瞬时杂交程度（即双链体覆盖率）、总探针覆盖率和离子强度。有趣的是，这些实验揭示了不同的动力学阶段，没有一个阶段与 Langmuir 动力学一致。初始阶段，双链体覆盖率仍然相对稀疏，表明有两种效应的融合：已形成的双链体阻止了靶分子与未杂交的探针结合，以及由于探针分子的消耗使固体支持物失活。这种解释与一种表面结构一致，其中未杂交的 MO 探针在 MO-DNA 双链体层下靠近固体支持物定位。随着双链体覆盖率的增加，只要没有先达到饱和，初始阶段就可以转变为一个不寻常的阶段，其特点是杂交速率对双链体覆盖率几乎没有依赖性，然后再延长达到平衡的时间。这些更为复杂的后期行为的可能起源正在被讨论。与已发表的 DNA 和肽核酸（PNA）探针数据进行比较，以寻找动力学的普遍趋势。这种比较揭示了在预期具有可比探针表面结构的情况下，存在定性相似性。此外，MO 单层被发现具有广泛的反应性，其范围跨越了已报道的 PNA 和 DNA 探针的范围。
