Compositions and Methods for Detecting Molecular Targets on Chromosomal DNA

Abstract

Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.

What is claimed is: 
     
         1 . A composition comprising: a nicking endonuclease fusion protein (NEFP) comprising the nicking endonuclease and a domain that binds to a constant region of an antibody. 
     
     
         2 . The composition according to  claim 1 , wherein the NEFP has at least 90% sequence identity to a sequence selected from the group consisting of: SEQ ID NOs: 5, 6, 7 and 8. 
     
     
         3 . The composition according to  claim 1 , wherein the NEFP is combined with a heparin salt in a buffer containing less than 100 mM NaCl. 
     
     
         4 . The composition according to  claim 1 , wherein the antibody binding domain is Protein A, Protein L or Protein G or a combination of one or more of Protein A, Protein L or Protein G in tandem. 
     
     
         5 . The composition according to  claim 1 , wherein the nicking endonuclease is Nt.CviPll or gHNH. 
     
     
         6 . The composition according to  claim 1 , wherein the NEFP further comprises a polymerase in a tripartite fusion or in a mixture. 
     
     
         7 . The composition according to  claim 1 , wherein the NEFP is immobilized on a substrate. 
     
     
         8 . The composition according to  claim 1 , wherein the NEFP is lyophilized. 
     
     
         9 . A kit comprising: the composition of  claim 1  and instructions for use. 
     
     
         10 . The kit according to  claim 9 , further comprising: a heparin salt in a low concentration inorganic salt buffer wherein the NEFP and the heparin may be lyophilized or in the buffer, in separate compartments or combined in the same compartment. 
     
     
         11 . The kit according to  claim 9 , wherein the NEFP has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7 and 8. 
     
     
         12 . The kit according to  claim 9 , further comprising a polymerase and one or more modified nucleotides and dNTPs. 
     
     
         13 . The kit according to  claim 12 , wherein the modified nucleotides are selected from the group consisting of d 5m CTP, biotinylated CTP and a dye labeled dNTP. 
     
     
         14 . A kit comprising: a nicking endonuclease fusion protein (NEFP) comprising the nicking endonuclease and a protein A or Protein G domain that binds to a constant region of an antibody; and a heparin salt in a low concentration inorganic salt buffer wherein the NEFP and the heparin may be lyophilized or in the buffer, in separate compartments or combined in the same compartment. 
     
     
         15 . A method for identifying a DNA location of a target protein that is on chromosomal DNA in a sample, comprising:
 (a) combining a nicking endonuclease fusion protein (NEFP) and a sample under conditions wherein the NEFP is reversibly prevented from directly binding to the chromosomal DNA; wherein
 i. the NEFP comprises a nicking endonuclease and a protein A or protein G domain; and 
 ii. a target protein bound to chromosomal DNA is present in the sample, wherein the target protein is directly or indirectly bound to a target protein-specific antibody, under conditions by which the protein A or protein G domain in the NEFP binds to the constant region of the target protein-specific antibody; 
   (b) allowing the nicking endonuclease that is bound to the target protein-specific antibody in (a) to nick the chromosomal DNA in a buffer containing magnesium or manganese ions for permitting the nicking activity of the NEFP;   (c) nick translating the nicked chromosomal DNA with a strand displacing polymerase in the presence of a mixture of dNTPs wherein one or more dNTPs in the mixture is modified for blocking secondary nicking;   (d) sequencing of the nick translated DNA produced in step (c) to identify the DNA location of the target protein on the chromosomal DNA.   
     
     
         16 . The method according to  claim 15 , wherein the target protein is a DNA binding protein. 
     
     
         17 . The method according to  claim 15 , wherein step (a) is done in a first buffer, wherein the first buffer contains a heparin and an inorganic salt. 
     
     
         18 . The method according to  claim 15 , wherein the sample is a cell sample. 
     
     
         19 . The method according to  claim 15 , wherein step (a) or a step prior to step (a) is done at a temperature in the range of 20° C.-100° C. 
     
     
         20 . The method according to  claim 15 , wherein the method is performed at a single temperature. 
     
     
         21 . The method according to  claim 15 , wherein step (a) further comprises washing away unbound NEFP and the heparin by replacing the first buffer with a second buffer containing an inorganic salt that is at a concentration that is higher than the concentration of the 
     
     
         22 . The method according to  claim 15 , wherein the NEFP comprises a nicking endonuclease fused to a Protein A or Protein G domain or any combination thereof in tandem. 
     
     
         23 . The method according to  claim 15 , wherein the nicking endonuclease in the NEFP is Nt.CviPll or gHNH. 
     
     
         24 . The method according to  claim 15 , wherein the one or more modified nucleotides are selected from the group consisting of d5mCTP, biotinylated CTP and a dye labeled dNTP. 
     
     
         25 . The method according to  claims 15 , wherein the method comprises immobilizing the nick translated DNA produced in step (c), before step (d). 
     
     
         26 . The method according to  claim 25 , further comprising preparing a library from the immobilized DNA. 
     
     
         27 . The method according to  claim 26 , comprising sequencing the library and identifying the location of the target protein in the DNA sequence. 
     
     
         28 . The method according to  claim 15 , wherein the target protein is diagnostic for cancer or treatment of cancer, the method further comprises determining the presence of the diagnostic target by microscopy and sequencing on the same cell sample or biopsy from a patient.