Acquisition of a Multimode Plate Reader - Project Summary We are requesting funds to purchase an Agilent BioTek Synergy Neo2 Hybrid Multi-Mode Micropla1e Reader as an equipment supplement for our Rl 5 grant which focuses on developing an understanding for the capabilities and limitationsofCRlSPR-dCas9 technology to target G-quadruplexes in promoter regions of several prominent genes in order to regulate their expression. We currently use a decade old,shared pla1e reader that has limited capabilities ( only fluorescence and absmption) and significantly lower resolution compared to a modern plate reader.Being a shared instrument, there are also significant constraints in terms of scheduling and access time for the current instrument, which was used by about ten different group last year. Synergy Neo2 instrument will be replacing this older instrument and provide us with on-demand access to a modem plate reader. The proposed work for the Rl 5 grant involves in vitro single molecule and bulk biophysical and biochemical studies in addition to in cellulo measurements to evaluate gene expression. Given the broad scope of the work, several different experimental approaches are used to characterize the nucleic acid constructs and their interactions with dCas9 and RNA polymerase. Such characterization and screerring efforts are critical before the more laborious in cellulo or single molecule studies are performed. The requested instrument will provide higher sensitivity for fluorescence and UV-Vis measuremen1s (monochromator-based detection), and it will enable us to perform fluorescence polarization, time resolved fluorescence and FRET (laser-based system), luminescence, and cell-based assays (with temperature and CO,/O2 control), which are not currently available. These additional capabilities and enhanced sensitivi1y will accelerate our efforts, reduce the demands on the required quantities of nucleic acid construc1s and provide alternative means to test the validity of results, such as fluorescence polarization serving as an alternative to gel-based binding assays. Based on the frequent use of the current plate reader by many groups, we anticipate this instrument to have a significant impact on the local research environment.
