Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine b...Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine by assembling T7 RNA polymerase on a small molecule-labeled DNA heteroduplex. The nanomachine, of which the RNA transcription activity can be quantitatively inhibited by protein binding, showed a great potential for small molecule-protein interaction assay. This finding enabled us to develop a novel homogeneous label-free strategy for assays of interactions between small molecules and their protein receptors. Three small molecule compounds and their protein receptors have been used to demonstrate the developed strategy. The results revealed that the protein-small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 64 nM with a detection limit of 0.2 nM. Due to its label-free, homogeneous, and fluorescence-based detection format, besides its desirable sensitivity this technique could be greatly robust, cost-efficient and readily automated, implying that the developed small molecule-protein interaction assay strategy might create a new methodology for developing intrinsically robust, sensitive and selective platforms for homogeneous protein detection.展开更多
基金supported by the National Natural Science Foundation of China (21025521, 21035001&20875027)the National Key Basic Re-search Program (2011CB911000)+3 种基金European Commission FP7-HEALTH-2010 Programme-GlycoHIT (260600)National Grand Program on Key Infectious Disease (2009ZX10004-312)Postdoctoral Science Foundation (20100480934) of ChinaChangjiang Scholars and Innovative Research Team in University Program and Natural Science Foundation of Hunan Province (10JJ7002)
文摘Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine by assembling T7 RNA polymerase on a small molecule-labeled DNA heteroduplex. The nanomachine, of which the RNA transcription activity can be quantitatively inhibited by protein binding, showed a great potential for small molecule-protein interaction assay. This finding enabled us to develop a novel homogeneous label-free strategy for assays of interactions between small molecules and their protein receptors. Three small molecule compounds and their protein receptors have been used to demonstrate the developed strategy. The results revealed that the protein-small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 64 nM with a detection limit of 0.2 nM. Due to its label-free, homogeneous, and fluorescence-based detection format, besides its desirable sensitivity this technique could be greatly robust, cost-efficient and readily automated, implying that the developed small molecule-protein interaction assay strategy might create a new methodology for developing intrinsically robust, sensitive and selective platforms for homogeneous protein detection.
