Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Nat Methods. 2011 Apr;8(4):335-40. doi: 10.1038/nmeth.1574. Epub 2011 Feb 20.
We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore-labeled DNA oligonucleotides to bind and unbind complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (for example, single-base-pair stepping of DNA translocases) along with the detection of properties of fluorescently labeled protein (for example, internal configuration).
我们提出了一种单分子仪器，它结合了分时超高分辨率双光阱与共焦荧光显微镜。在一个演示实验中，我们观察到单个荧光标记的 DNA 寡核苷酸与悬浮在两个捕获珠之间的互补 DNA 结合和解离。与单荧光检测同时，我们清楚地观察到连接延伸的毫微微米级变化。荧光读出允许我们确定双链体熔解速率作为力的函数。新仪器将能够同时测量单个 DNA 结合蛋白的毫微微米级机械运动（例如，DNA 转位酶的单个碱基对步移），同时检测荧光标记蛋白的性质（例如，内部结构）。
