One month Exchange Program Report
Host Organization: Kyoto University, Japan
Training Supervisor: Professor Motonari UESUGI
Screening for Self-Assembly Compounds from the single hits of ANS and Nile Red
Fudan University, School of Pharmacy
2nd Year in Ph.D. Course
Self-Assembly in classic sense can be defined as spontaneous and reversible organization of small molecular units into an ordered structure by non-covalent interaction. By self-assembling, the small molecular can form different morphologies. With the development of optical microscope and other measurement methods, it can test the morphology of the self-assembly molecular by using the different dyes such as ANS, Nile Red, DCVJ, Thioflavin T, Congo Red and so on1. Different Self-Assembly molecular has been used in tuning biological function2-5.
Based on the previous work that has been finished in Uesugi’s Lab. They screened self-assembly compounds through environment-responsonsive probes ANS and Nile Red. From the 8000 in-house compounds, they obtained 116 dual hits which can respond both probes ANS and Nile Red, and 558 single hit which could respond either probe ANS or Nile Red. So in our present work, we want to screen for Self-Assembly compounds from the single hits of ANS and Nile Red, Which can respond for the both dyes Thioflavin T and Lipidye or one of them.
From the first screen of the self-assembly assay, we got too much hits for Thioflavin T dye and Lipidye. But for Thioflavin T dye of the plates 13-17, when incubated for overnight, and the self-assembly hits is more reasonable (Table 1). So we did the second screen for the two dyes, after we measured the fluorescence, we found incubation time is more important for Thioflavin T dye but not sure for the lipidye.
|Plate No.||Incubation time||Compounds No.||Hits No.||Ratio|
|Thioflavin T dye||Plates 1-12||2 hours||1360||277||20%|
After the first round screening of the single hits, I selected the front 1-80 hits of the two dyes to subject the second screen. The incubation time was prolonged to overnight. After we analyzed the results of the second screening for the front 1-80 hits of the two dyes. We got the top 10 hits of the two dyes and the results are shown as follows.
The fluorescence was measured on the instrument microplate reader using Tecan i-control software. 100μL PBS was added to the 96-well plate, and then added 1μL 5mM single hit to the plate according to the hit number. After all the negative control and positive control was added, put the plate into the plate shaker and shake for 1 min at the speed of 80r/min. Then measure the background fluorescence and add 1μL corresponding dye to the working plate. After 30min incubation, measure the fluorescence at the corresponding wavelength. After all the measurement was finished, we evaluated the compounds whether the compound can be selected as hit according to the negative control and positive control.
This work was supported by JSPS A3 Foresight Program : Asian Chemical Probe Research Hub. Thank you for Prof. Uesugi and Ms. Hue for their precious suggestion and directions. Thank you all the members in Uesugi’s Lab.
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