Compositions comprising resurfaced cell-penetrating nanobodies and methods of use thereof

Abstract

The disclosure encompasses resurfaced cell-penetrating nanobodies and their methods of use. The resurfacing of nanobodies with positively-charged amino acids facilitates their penetration into a cell and allows targeting of a specific intracellular protein.

What is claimed is: 
     
         1 . A method of binding a target protein, the method comprising:
 providing a cell-penetrating nanobody, having binding affinity for the target protein and comprising a modified framework region, wherein the modified framework region has one or more amino acid residues that are substituted as compared to a framework region of a wild-type nanobody and the modified framework region has an exposed polycationic surface; and   contacting the target protein with the cell-penetrating nanobody in the cytosol of a cell.   
     
     
         2 . The method of  claim 1 , wherein the one or more amino acid residues are substituted with arginine or lysine as compared to the framework region of the wild-type nanobody. 
     
     
         3 . The method of  claim 1 , wherein the modified framework region has at least nine amino acid residues that are substituted with arginine or lysine as compared to the framework region of the wild-type nanobody. 
     
     
         4 . The method of  claim 1 , wherein the cell-penetrating nanobody has a theoretical net charge ranging from about +10 to about +35. 
     
     
         5 . The method of  claim 4 , wherein the theoretical net charge ranges from about +10 to about +20. 
     
     
         6 . The method of  claim 1 , wherein the cell-penetrating nanobody is fused to a reporter protein. 
     
     
         7 . The method of  claim 6 , wherein the reporter protein is chosen from: a superpositive green fluorescent protein GFP (spGFP), a superpositive far-red fluorescent protein (sp-mNeptune), a supernegative green fluorescent protein (snGFP), and a supernegative far-red fluorescent protein (sn-mNeptune). 
     
     
         8 . The method of  claim 1 , wherein the modified framework region comprises the framework protein sequence selected from the group consisting of pcNB1, pcNB2, and pcNB3. 
     
     
         9 . The method of  claim 1 , wherein the framework region of the wild-type nanobody comprises a framework protein sequence selected from the group consisting of NB1, NB2, and NB3. 
     
     
         10 . A method of detecting a target protein, the method comprising:
 providing a cell-penetrating nanobody; and   contacting the target protein with the cell-penetrating nanobody in the cytosol of a cell,   wherein the cell-penetrating nanobody comprises a modified framework, wherein the modified framework region has one or more amino acid residues that are substituted as compared to a framework region of a wild-type nanobody and the modified framework region has an exposed polycationic surface,   wherein the cell-penetrating nanobody has binding affinity for the target protein, and   wherein the cell-penetrating nanobody is fused to a reporter protein.   
     
     
         11 . The method of  claim 10 , wherein the one or more amino acid residues are substituted with arginine or lysine as compared to the framework region of the wild-type nanobody. 
     
     
         12 . The method of  claim 10 , wherein the modified framework region has at least nine amino acid residues that are substituted with arginine or lysine as compared to the framework region of the wild-type nanobody. 
     
     
         13 . The method of  claim 10 , the cell-penetrating nanobody having a theoretical net charge ranging from about +10 to about +35. 
     
     
         14 . The method of  claim 13 , wherein the theoretical net charge ranges from about +10 to about +20. 
     
     
         15 . The method of  claim 10 , wherein the reporter protein is chosen from: a superpositive green fluorescent protein GFP (spGFP), a superpositive far-red fluorescent protein (sp-mNeptune), a supernegative green fluorescent protein (snGFP), and a supernegative far-red fluorescent protein (sn-mNeptune). 
     
     
         16 . The method of  claim 10 , wherein modified framework region comprises the framework protein sequence selected from the group consisting of pcNB1, pcNB2, and pcNB1. 
     
     
         17 . The method of  claim 10 , wherein the framework region of the wild-type nanobody comprises a framework protein sequence selected from the group consisting of NB1, NB2, and NB3.