FRET-labeled siRNA probes for tracking assembly and disassembly of siRNA nanocomplexes.
FRET-labeled siRNA probes for tracking assembly and disassembly of siRNA nanocomplexes.
复制标题
DOI：
10.1021/nn3013838
复制
发表时间：
2012-07-24
期刊：
影响因子：
17.1
通讯作者：
Anderson, Daniel G.
中科院分区：
文献类型：
--
作者：
Alabi, Christopher A.;Love, Kevin T.;Sahay, Gaurav;Stutzman, Tina;Young, Whitney T.;Langer, Robert;Anderson, Daniel G.
The assembly, stability and timely disassembly of short interfering RNA (siRNA) nanocomplexes all have the potential to affect the efficiency of siRNA delivery and gene silencing. As such, the design of new probes that can measure these properties without significantly perturbing the nanocomplexes or their environment may facilitate the study and further development of new siRNA nanocomplexes. Herein, we study Förster resonance energy transfer (FRET)-labeled siRNA probes that can track the assembly, stability and disassembly of siRNA nanocomplexes in different environments. The probe is composed of two identical siRNAs, each labeled with a fluorophore. Upon nanocomplex formation, the siRNA-bound fluorophores become locally aggregated within the nanocomplex and undergo FRET. A key advantage of this technique is that the delivery vehicle (DV) need not be labeled, thus enabling the characterization of a large variety of nanocarriers, some of which maybe difficult or even impossible to label. We demonstrate proof-of-concept by measuring the assembly of various DVs with siRNAs and show good agreement with gel electrophoresis experiments. As a consequence of not having to label the DV, we are able to determine nanocomplex biophysical parameters such as the extracellular apparent dissociation constants (KD) and intracellular disassembly half-life for several in-house and proprietary commercial DV’s. Furthermore, the lack of DV modification allows for a true direct comparison between DVs as well as correlation between their biophysical properties and gene silencing.
登录
查看更多内容
影响因子：
3.9
作者：
Jiang, Shan;Zhang, Yong
通讯作者：
Zhang, Yong
影响因子：
2.9
作者：
Raemdonck, Koen;Remaut, Katrien;De Smedt, Stefaan C.
通讯作者：
De Smedt, Stefaan C.
影响因子：
3.4
作者：
Eastman, SJ;Siegel, C;Scheule, RK
通讯作者：
Scheule, RK
影响因子：
2.9
作者：
Saminathan, M;Antony, T;Thomas, TJ
通讯作者：
Thomas, TJ
影响因子：
12.4
作者：
Akinc, Akin;Goldberg, Michael;Anderson, Daniel G.
通讯作者：
Anderson, Daniel G.