In vivo detection of phospholipase activation
The present invention is directed to a phospholipid-based NIR molecular beacon, having a phospholipid moiety; with an NIR fluorophore moiety covalently linked to a phospholipid glycerol backbone and a quencher moiety covalently linked to the phospholipid glycerol backbone. Additionally, provided herein is methods of analyzing a sample for the presence of a phospholipase and methods of identifying the activity of a phospholipase in vivo utilizing phospholipid-based NIR molecular beacon.
1. A method of analyzing a sample for the presence of a phospholipase, the method comprising:
contacting a sample suspected of comprising a phospholipase with a phospholipid-based near infrared (NIF) molecular beacon comprising (i) a phospholipid moiety with a glycerol backbone; (ii) a first fluorophore moiety covalently linked to the glycerol backbone either directly or via a linker, wherein said first fluorophore moiety is an NIR fluorophore moiety; and (iii) a fluorescence quencher moiety covalently linked to said phospholipid glycerol backbone either directly or via a linker, wherein the fluorophore moiety, the fluorescence quencher moiety or both are uncharged when covalently linked at either or both sn-1 or sn-2 fatty acyl positions; and
detecting the NIR fluorescence of said NIR fluorophore moiety as a function of time, wherein an increase in the NIR fluorescence as a function of time correlates with the presence of the phospholipase in the sample.
2. The method of claim 1 , wherein said NIR fluorophore is covalently bound to the glycerol backbone at either the sn-1 or sn-2 fatty acyl positions or to the sn-3 head group.
3. The method of claim 2 , wherein said quencher moiety is a second fluorophore covalently bound to the remaining unbound fatty acyl position or the sn-3 head group.
4. The method of claim 3 , wherein said second fluorophore moiety or quencher moiety is covalently linked to the phospholipid moiety either directly or via a linker.
5. The method of claim 1 , wherein said sample is a tumor cell or an extract of a tumor cell.
6. The method of claim 1 , wherein said sample is a cultured cell, a primary cell culture or their combination.
7. A method of monitoring the activity of a phospholipase in vivo, comprising the steps of:
exposing a tissue to a phospholipid-based near infrared (NIR) molecular beacon under conditions effective to permit said phospholipase to cleave said beacon, wherein said beacon comprises (i) a phospholipid moiety with a glycerol backbone; (ii) a first fluorophore moiety covalently linked to the glycerol backbone either directly or via a linker, wherein said first fluorophore moiety is an NIR fluorophore moiety; and (iii) a fluorescence quencher moiety covalently linked to said phospholipid glycerol backbone either directly or via a linker, wherein the fluorophore moiety, the fluorescence quencher moiety or both are uncharged when covalently linked at either or both sn-1 or sn-2 fatty acyl positions; and
detecting the fluorescence of said fluorophore moiety as a function of time, wherein an increase in the quantity or rate of accumulation of fluorescence as compared to a control reaction identifies the activity of the phospholipase.
8. The method of claim 7 , wherein said NIR fluorophore is covalently bound to the glycerol backbone at either the sn-1 or sn-2 fatty acyl positions or to the sn-3 head group.
9. The method of claim 8 , wherein the quencher moiety is a second fluorophore covalently bound to the remaining unbound fatty acyl position or the sn-3 head group.
10. The method of claim 9 , wherein said second fluorophore moiety or quencher moiety is covalently linked to the phospholipid moiety either directly or via a linker.