Annexin-based apoptosis markers

Abstract

The present invention describes an annexin derivative and a method of using the annexin derivative as a biosensor for real-time visualization of phosphatidylserine exposure, apoptosis, live-cell imaging and monitoring of cell health.

1 - 14 . (canceled) 
     
     
         15 . A method of detecting apoptosis, comprising:
 providing an annexin derivative comprising: an annexin comprising one or more amino acids conjugated to a polarity sensitive fluorophore, wherein the one or more amino acids were inserted into or substituted at a polarity changing site on the annexin;   contacting the annexin derivative to a sample comprising cells; and   detecting a fluorescence in the sample.   
     
     
         16 . The method of  claim 15 , wherein the annexin is a cysteine less or lysine less variant prior to the insertion or substitution of the one or more amino acids at the polarity changing site, or a cysteine or lysine is deleted or substituted away from a wild-type annexin during the insertion or substitution of the one or more amino acids at the polarity changing site. 
     
     
         17 . The method of  claim 15 , wherein the polarity changing site on the annexin is a membrane-interaction site on the annexin or a site that undergoes a conformational change. 
     
     
         18 . The method of  claim 15 , wherein the polarity changing site is a loop region on the annexin. 
     
     
         19 . The method of  claim 15 , wherein the annexin is selected from the group consisting of annexins A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, All, A13, ci0100146873, ci0100136930, ci0100137443, ci0100153687, and ci0100138049, B9, B10, B11, B13, nex-1, nex-2, nex-3, nex-4, C1, C2, D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, annexins from  Schistosoma mansoni,  and annexins from  Giardia lamblia.    
     
     
         20 . The annexin derivative of  claim 15 , wherein the annexin is annexin B12. 
     
     
         21 . The annexin derivative of  claim 20 , wherein the one or more amino acids were inserted next to or substituted at or around residues 26-33, 70-73, 97-105, 141-145, 180-189, 226-229, 256-264, and/or 301-304. 
     
     
         22 . The method of  claim 20 , wherein the one or more amino acids were inserted next to or substituted at or around a residue at position 101, 260 or both. 
     
     
         23 . The method of  claim 15 , wherein the annexin is annexin A5. 
     
     
         24 . The method of  claim 23 , wherein the one or more amino acids were inserted next to or substituted at or around a residue at position 262. 
     
     
         25 . The method of  claim 15 , wherein the one or more amino acids is cysteine. 
     
     
         26 . The method of  claim 15 , wherein detecting the fluorescence comprises detecting an increase of fluorescence intensity and/or a shift in the wavelength of the fluorescence. 
     
     
         27 . The method of  claim 26 , wherein the increase of fluorescence intensity is one or more orders of magnitude. 
     
     
         28 . The method of  claim 26 , wherein the polarity sensitive fluorophore is N,N′-Dimethyl-N-(iodoacetyl)-N′(7-nitrobenz-2-oxa-1,2-diazol-4-yl)ethylenediamine (“IANBD”) and the fluorescence is detected at a λ max  of about 500 to about 600 nm; or the polarity sensitive fluorophore is 6-Bromoacetyl-2-dimethylaminonaphthalene (“BADAN”) and the fluorescence is detected in the blue light range. 
     
     
         29 . The method of  claim 28 , wherein detecting is performed in real time and/or performed in a high-throughput screening system. 
     
     
         30 . A method of monitoring cell health, comprising:
 providing an annexin derivative comprising: an annexin comprising one or more amino acids conjugated to a polarity sensitive fluorophore, wherein the one or more amino acids were inserted into or substituted at a polarity changing site on the annexin;   contacting the annexin derivative to a sample comprising cells; and   monitoring the fluorescence, wherein an increase in the fluorescence intensity and/or a shift in the wavelength of the fluorescence indicate that a cell is undergoing apoptosis and an absence of an increase in the fluorescence intensity and/or an absence of a shift in the wavelength of the fluorescence indicate that a cell is healthy.   
     
     
         31 - 45 . (canceled)