Lehigh University, Department of Chemistry, 6 E. Packer Ave, Bethlehem, PA 18015, USA.
Nanoscale. 2011 Feb;3(2):581-91. doi: 10.1039/c0nr00479k. Epub 2010 Nov 19.
We applied the combined approach of evanescent nanometry and force spectroscopy using magnetic tweezers to quantify the degree of hybridization of a single synthetic single-stranded DNA oligomer to a resolution approaching a single-base. In this setup, the 200 nucleotide long DNA was covalently attached to the surface of an optically transparent solid support at one end and to the surface of a superparamagnetic fluorescent microsphere (force probe) at the other end. The force was applied to the probes using an electromagnet. The end-to-end molecular distance (i.e. out-of-image-plane position of the force probe) was determined from the intensity of the probe fluorescence image observed with total-internal reflectance microscopy. An equation of state for single stranded DNA molecules under tension (extensible freely jointed chain) was used to derive the penetration depth of the evanescent field and to calibrate the magnetic properties of the force probes. The parameters of the magnetic response of the force probes obtained from the equation of state remained constant when changing the penetration depth, indicating a robust calibration procedure. The results of such a calibration were also confirmed using independently measured probe-surface distances for probes mounted onto cantilevers of an atomic force microscope. Upon hybridization of the complementary 50 nucleotide-long oligomer to the surface-bound 200-mer, the changes in the force-distance curves were consistent with the quantitative conversion of 25% of the original single-stranded DNA to its double-stranded form, which was modeled as an elastic rod. The method presented here for quantifying the hybridization state of the single DNA molecules has potential for determining the degree of hybridization of individual molecules in a single molecule array with high accuracy.
我们采用消逝波纳米测量和磁力镊力谱学相结合的方法，以单碱基分辨率定量测量单链合成 DNA 寡聚物的杂交程度。在该设置中，200 个核苷酸长的 DNA 一端共价连接到光学透明固体基底的表面，另一端连接到超顺磁荧光微球（力探针）的表面。使用电磁铁对探针施加力。使用全内反射显微镜观察探针荧光图像的强度来确定分子的末端到末端距离（即力探针的离焦位置）。使用拉伸状态下单链 DNA 分子的状态方程（可自由伸缩的连接链）来推导消逝场的穿透深度并校准力探针的磁性。从状态方程获得的力探针磁响应参数在改变穿透深度时保持不变，表明校准程序稳健。使用独立测量的安装在原子力显微镜悬臂上的探针表面距离也证实了这种校准的结果。当互补的 50 个核苷酸长的寡聚体与表面结合的 200 -mer 杂交时，力-距离曲线的变化与 25%的原始单链 DNA定量转化为双链形式一致，这被建模为弹性棒。这里提出的用于定量单 DNA 分子杂交状态的方法具有以高精度确定单分子阵列中各个分子的杂交程度的潜力。
