A force sensor that converts fluorescence signal into force measurement utilizing short looped DNA.
A force sensor that converts fluorescence signal into force measurement utilizing short looped DNA.
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DOI：
10.1016/j.bios.2018.08.073
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发表时间：
2018-12-15
影响因子：
12.6
通讯作者：
Balci H
中科院分区：
文献类型：
--
作者：
Mustafa G;Chuang CY;Roy WA;Farhath MM;Pokhrel N;Ma Y;Nagasawa K;Antony E;Comstock MJ;Basu S;Balci H
提出了一种力传感器的概念，其中荧光信号通过单分子Förster共振能量转移（smFRET）转换为力信息。传感器的基本设计是~100个碱基对（bp）长的双链DNA（dsDNA），其被桥接其末端的核酸二级结构（NAS）限制为环状构象。环状的dsDNA在NAS上产生张力，当张力足够高时将其展开。在NAS上放置的供体和受体（D&A）荧光团之间的FRET效率报告其折叠状态。通过DNA发夹桥接具有不同长度的三个dsDNA构建体，并且滴定KCl以改变所施加的力。在这些原理验证测量之后，使用dsDNA构建体之一来维持由凝血酶结合适体（TBA）在张力下形成的G-四链体（GQ）构建体，同时其与去稳定化蛋白质和稳定化小分子相互作用。用高分辨率光镊（OT）测量独立地研究了展开TBA-GQ所需的力，其确定相关力为几pN，这与环状dsDNA产生的力一致。所提出的方法是特别有前途的，因为它能够使用高度平行的基于FRET的测定来研究NAS、蛋白质和小分子的相互作用，同时NAS保持在近似恒定的力下。
A force sensor concept is presented where fluorescence signal is converted into force information via single-molecule Förster resonance energy transfer (smFRET). The basic design of the sensor is a ~100 base pair (bp) long double stranded DNA (dsDNA) that is restricted to a looped conformation by a nucleic acid secondary structure (NAS) that bridges its ends. The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough. The FRET efficiency between donor and acceptor (D&A) fluorophores placed across the NAS reports on its folding state. Three dsDNA constructs with different lengths were bridged by a DNA hairpin and KCl was titrated to change the applied force. After these proof-of-principle measurements, one of the dsDNA constructs was used to maintain the G-quadruplex (GQ) construct formed by thrombin binding aptamer (TBA) under tension while it interacted with a destabilizing protein and stabilizing small molecule. The force required to unfold TBA-GQ was independently investigated with high-resolution optical tweezers (OT) measurements that established the relevant force to be a few pN, which is consistent with the force generated by the looped dsDNA. The proposed method is particularly promising as it enables studying NAS, protein, and small molecule interactions using a highly-parallel FRET-based assay while the NAS is kept under an approximately constant force.
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影响因子：
14.9
作者：
Le TT;Kim HD
通讯作者：
Kim HD
影响因子：
3.8
作者：
Mathias, Jordan;Okyere, Robert;Kankia, Besik
通讯作者：
Kankia, Besik
DOI：
10.1073/pnas.0511214103
发表时间：
2006-02-21
影响因子：
11.1
作者：
Moiseev, L;Ünlü, MS;Cantor, CR
通讯作者：
Cantor, CR
影响因子：
15
作者：
Punnoose, Jibin Abraham;Cui, Yunxi;Mao, Hanbin
通讯作者：
Mao, Hanbin
DOI：
10.1007/978-1-4614-0980-9_24
发表时间：
2012-01-01
期刊：
VIRAL MOLECULAR MACHINES
影响因子：
--
作者：
Chemla, Yann R.;Smith, Douglas E.
通讯作者：
Smith, Douglas E.