The Fluorogen Activation Modules (the "FAMs")
Sharp Edge is bringing a new class of targeted biosensors to drug discovery and trafficking biology. Fluorogenic Activating Modules (or FAMs)* provide a more accurate and detailed detection method for cellular trafficking that is amendable to high-throughput screening and more detailed follow-up.
How the FAMs work
The FAMs are single-chain antibody fragments (scFvs) of approximately 140 amino acids that have been selected to bind and activate the fluorescence of specially designed dyes (the Flurogens).(1)
FAM-fusions are created that behave similarly to GFP-tagged proteins. However, there are several advantages to the FAM technology.
The ability to genetically target the FAM by creating fusion proteins (Targeting), combined with the ability to modulate the properties of the Flurogens (Sensing)) leads to a variety of new types of assays. For example, Sharp Edge Labs has developed a new set of cell-surface protein trafficking assays that are more detailed, and yet simpler than competing technologies.
Trafficking: Targeting + Sensing
By targeting the FAM to a cell surface protein (a GPCR for eaxample), and using cell impermenant and cell permeant dyes, we've created an easy and direct assay for receptor internalization.
Note that se-Red-xc is excluded from the cytoplasm, and therefor provides nicely selective labelling for the plasma membrane component of the target protein. Note also that unlike a surface ELISA, no washing is necessary because the Fluorogen is dark until it binds the FAM. The figure below shows this comparison in a live cell assay.
se-Red-s labels both surface protein and protein in the biosynthetic pathway (the ER/Golgi in this figure) and measures total protien, similar to GFP. The ability to switch dyes (and even add se-Red-S after imaging with se-Red-xc) provides and easy way to untangle cell-surface from total protein, which is difficult with other labelling methods.
A variety of fluorogenic sensor dyes has also been created, allowing targeted sensing, which creates a powerful tool for understanding trafficking. By using an extracellular pH Sensing Fluorogen, you can get a clean and uniqe signal from surface protein that has entered the endocytic pathway.
In this case, the pH sensor dye changes "color" when it moves from the neutral environment of the extracellular space, to the acidic pH of the endosome. Note that the sensor dye only reads out the pH at the location of the FAP, since it is dark otherwise (that is, it is a true, targeted sensor). This assay also requires no washing, and is amenable to live-cell and fixed cell preparations.
These features are especially useful for studying trafficking of well-known drug targets such as GPCRs, Ion Channels, and Transporters.
*The FAMs are covered by US Patent 8,664,364 and other US and international pending patent applications.
(1) Szent-Gyorgyi C, Schmidt BF, Creeger Y, Fisher GW, Zakel KL, Adler S, Fitzpatrick JA, Woolford CA, Yan Q, Vasilev KV, Berget PB, Bruchez MP, Jarvik JW, Waggoner A. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nature biotechnology. 2008;26(2):235-40. Epub 2007/12/25. doi: 10.1038/nbt1368. PubMed PMID: 18157118.
(2) Fitzpatrick JA, Yan Q, Sieber JJ, Dyba M, Schwarz U, Szent-Gyorgyi C, Woolford CA, Berget PB, Waggoner AS, Bruchez MP. STED nanoscopy in living cells using Fluorogen Activating Proteins. Bioconjugate chemistry. 2009;20(10):1843-7. Epub 2009/01/01. doi: 10.1021/bc900249e. PubMed PMID: 20976031; PubMed Central PMCID: PMC2957894
(3) Szent-Gyorgyi C, Schmidt BF, Fitzpatrick JA, Bruchez MP. Fluorogenic Dendrons with Multiple Donor Chromophores as Bright Genetically Targeted and Activated Probes. Journal of the American Chemical Society. 2010;132:6.