School of Medicine and Surgery, Università di Milano-Bicocca, Monza (MB), Italy.
Department of Physics, Emory University, Atlanta, Georgia.
Biophys J. 2019 Mar 5;116(5):760-771. doi: 10.1016/j.bpj.2019.01.027. Epub 2019 Feb 1.
2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.
2,6-二氨基嘌呤（DAP）是腺嘌呤的核苷类似物。当它被掺入双链 DNA（dsDNA）时，它与胸腺嘧啶形成三个氢键。DAP 在自然界中很少见，其取代会改变 DNA 分子的物理特性，而不会牺牲序列特异性。在这里，我们表明，除了稳定双链杂交外，DAP 取代还会改变 dsDNA 的力学和构象特性。热融解实验表明，DAP 取代会提高融解温度，而不会降低序列依赖性效应。我们使用原子力显微镜（AFM）、磁镊（MT）纳米力学测定和圆二色性光谱的组合，证明 DAP 取代会增加 dsDNA 的弯曲刚性，但也会促进构象转变，表现为分子长度的变化。DAP 取代会增加 DNA 的静态和动态持久长度（分别通过 AFM 和 MT 测量）。在静态情况下（AFM），即不给分子施加张力时，DAP-DNA 的轮廓长度看起来比野生型（WT）-DNA 短；在张力下（MT），它们具有相似的动态轮廓长度。在超过 60 pN 的张力下，WT-DNA 会由于链分离（张力诱导的融解）和自发采用一种称为 S-DNA 的构象而经历特征性的过度拉伸。WT-DNA 在接近零加载速率下的循环过度拉伸和松弛通常会产生滞后，表明张力诱导的融解；相反，DAP-DNA 的循环拉伸几乎没有或没有滞后，与 S 形的采用一致，类似于已报道的富含 GC 的序列。然而，DAP-DNA 的过度拉伸与富含 GC 的过度拉伸不同，它发生在明显较低的张力下。在生理盐条件下，均匀混合的 AT/GC DNA 通常在 60 pN 左右过度拉伸。富含 GC 的序列在相似甚至略高的张力下过度拉伸。在这里，我们表明 DAP-DNA 在 52 pN 处过度拉伸。总之，DAP 取代降低了 B 型双螺旋的整体稳定性，在零张力下偏向于非 B 型 DNA 螺旋构象，并在高张力下促进 B 到 S 的转变。
