My concept for spatial multiplexing enables the use of many fluorescent protein (FP) biosensors, enables the use of affinity series of biosensors, can be used with enhancer-promoter reporters, and will enable addressing and quantification of the telomere repeat length of all 92 human diploid telomeres. The current record for live cell multiplexing biosensors is 4plex by Piljic and Schultz of protein kinase C, CaMIIa, Annexin A4 and Ca++ ions using plasma membrane YFP/CFP ratio (PM-CKAR,), cytosol YFP/CFP ratio (CY-CaMIIa), mCherry/mOrange (Annexin A4) and Fura Red (Ca++ ions, respectively, My approach, called Tattletales, started with the observation by Robinett et al, who localized 512 GFP-nls-LacI to a 256 LacO array as a 200 nm diameter diffraction limited spot. Many DNA binding proteins, of known sequence specifities, exist (LacI, TetR, GalR, ZFN's and ZF-FPs, TALEN's and TALE-FPs) and can be fused (as cDNAs) to different fluorescent proteins and FP biosensors. Many biosensors are available as affinity series. I realized that spatial multiplexing of many DNA binding protein-reporters by localizing to different spots in the cell nucleus and distinguished by combinatorial addressing. When using cyan-yellow FRET biosensors, and blue, red and far-red FP's enable 23 = 8 addresses. Adding orange and very-far-red (mNeptune) enable 25=32 addresses. Switching to single FP biosensors (ex. GCaMP6 for Ca++) will enable more addresses (26=64), higher signal-to-noise ratio and simplify experiments. Fluorescence nanoscopes will result in better spatial and temporal resolution, enabling use of even more simultaneous biosensors. I have released Tattletales to the public domain.
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