Efficient synthesis of protein-oligonucleotide conjugates

Abstract

The present invention relates to an improved method for forming a protein-oligonucleotide conjugate. The method is particularly amenable for forming antibody-oligonucleotide conjugates. The invention further concerns the conjugate molecules produced using such improved methods.

1 . A method for producing an oligonucleotide-protein conjugate, wherein said method comprises conjugating an oligonucleotide-heterofunctional linker conjugate to a protein having a thiolated group formed by thiolation of an amino group of said protein; 
 wherein:    said linker is conjugated to said oligonucleotide by a chemical bond formed between an atom of a first group of said linker and an amino group of said oligonucleotide, and wherein said linker possesses a second group that is reactive with said thiolated group of said protein and forms a chemical bond therewith.    
     
     
         2 . The method of  claim 1 , wherein said amino group of said oligonucleotide is at the 3′ end of said oligonucleotide.  
     
     
         3 . The method of  claim 2 , wherein said method additionally comprises forming said oligonucleotide having said 3′ amino group.  
     
     
         4 . The method of  claim 2 , wherein said oligonucleotide having said 3′ amino group is formed by synthesizing said oligonucleotide on a 3 40  -amino CPG solid support.  
     
     
         5 . The method of  claim 1 , wherein said amino group of said oligonucleotide is at the 5′ end of said oligonucleotide.  
     
     
         6 . The method of  claim 1 , wherein said method additionally comprises forming said oligonucleotide having said 5′ amino group.  
     
     
         7 . The method of  claim 1 , wherein said amino group of said oligonucleotide is at an internal site of said oligonucleotide.  
     
     
         8 . The method of  claim 7 , wherein said method additionally comprises forming said oligonucleotide having said internal amino group.  
     
     
         9 . The method of  claim 1 , wherein said modified amino group is C7 CPG.  
     
     
         10 . The method of  claim 1 , wherein said first group of said heterofunctional linker is an NHS group.  
     
     
         11 . The method of  claim 1 , wherein said second group of said heterofunctional linker is a maleimide group.  
     
     
         12 . The method of  claim 1 , wherein said heterofunctional linker is selected from the group consisting of Sulfo-SMCC; Sulfo-EMCS; Sulfo-GMBS; Sulfo-KMUS; Sulfo-MBS; Sulfo-SIAB; Sulfo-SMPB; Sulfo-LC-SMPT; SVSB; SIACX; SIA, SIAXX; and NPIA.  
     
     
         13 . The method of  claim 12 , wherein said heterofunctional linker is sulfo-SMCC.  
     
     
         14 . The method of  claim 1 , wherein said thiolated group of said protein is derived from an iminothiolane moiety.  
     
     
         15 . The method of  claim 1 , wherein said method additionally comprises forming said protein having said thiolated group.  
     
     
         16 . The method of  claim 15 , wherein said thiolated group is formed by reacting the amino group of a protein with iminothiolane.  
     
     
         17 . The method of  claim 1 , wherein said protein is an enzyme, hapten, immunoglobulin, streptavidin, avidin, or a phycobillin protein.  
     
     
         18 . The method of  claim 17 , wherein said protein is an enzyme.  
     
     
         19 . The method of  claim 18 , wherein said enzyme is selected from the group consisting of alkaline phosphatase, β-galactosidase, horse radish peroxidase, and urease.  
     
     
         20 . The method of  claim 17 , wherein said protein is a hapten.  
     
     
         21 . The method of  claim 17 , wherein said protein is an immunoglobulin.  
     
     
         22 . The method of  claim 21 , wherein said immunoglobulin is an immunoglobulin that is able to bind to a drug, a receptor, a receptor ligand, or a tumor antigen.  
     
     
         23 . The method of  claim 21 , wherein said immunoglobulin is able to bind an antigen that is characteristic of a pathogen.  
     
     
         24 . The method of  claim 23 , wherein said pathogen is a virus.  
     
     
         25 . The method of  claim 24 , wherein said pathogen is a bacteria or fungus.  
     
     
         26 . The method of  claim 17 , wherein said protein is a streptavidin protein.  
     
     
         27 . The method of  claim 17 , wherein said protein is an avidin protein.  
     
     
         28 . The method of  claim 17 , wherein said protein is a phycobillin protein.  
     
     
         29 . An oligonucleotide-protein conjugate produced through the process comprising conjugating an oligonucleotide-heterofunctional linker conjugate to a protein having a thiolated group formed by thiolation of an amino group of said protein; 
 wherein:    said linker is conjugated to said oligonucleotide by a chemical bond formed between an atom of a first group of said linker and an amino group of said oligonucleotide, and wherein said linker possesses a second group that is reactive with said thiolated group of said protein and forms a chemical bond therewith.    
     
     
         30 . The oligonucleotide-protein conjugate of  claim 29 , wherein said amino group of said oligonucleotide is at the 3′ end of said oligonucleotide.  
     
     
         31 . The oligonucleotide-protein conjugate of  claim 29 , wherein said amino group of said oligonucleotide is at the 5′ end of said oligonucleotide.  
     
     
         32 . The oligonucleotide-protein conjugate of  claim 29 , wherein said amino group of said oligonucleotide is at an internal site of said oligonucleotide.  
     
     
         33 . The oligonucleotide-protein conjugate of  claim 29 , wherein said protein is an enzyme, hapten, immunoglobulin, streptavidin, avidin, or a phycobillin protein.  
     
     
         34 . The oligonucleotide-protein conjugate of  claim 33 , wherein said protein is an enzyme.  
     
     
         35 . The oligonucleotide-protein conjugate of  claim 34 , wherein said enzyme is selected from the group consisting of alkaline phosphatase, β-galactosidase, horse radish peroxidase, and urease.  
     
     
         36 . The oligonucleotide-protein conjugate of  claim 33 , wherein said protein is a hapten.  
     
     
         37 . The oligonucleotide-protein conjugate of  claim 33 , wherein said protein is an immunoglobulin.  
     
     
         38 . The oligonucleotide-protein conjugate of  claim 37 , wherein said immunoglobulin is an immunoglobulin that is able to bind to a drug, a receptor, a receptor ligand, or a tumor antigen.  
     
     
         39 . The oligonucleotide-protein conjugate of  claim 37 , wherein said immunoglobulin is able to bind an antigen that is characteristic of a pathogen.  
     
     
         40 . The oligonucleotide-protein conjugate of  claim 39 , wherein said pathogen is a virus.  
     
     
         41 . The oligonucleotide-protein conjugate of  claim 39 , wherein said pathogen is a bacteria or fungus.  
     
     
         42 . The oligonucleotide-protein conjugate of  claim 33 , wherein said protein is a streptavidin protein.  
     
     
         43 . The oligonucleotide-protein conjugate of  claim 33 , wherein said protein is an avidin protein.  
     
     
         44 . The oligonucleotide-protein conjugate of  claim 33 , wherein said protein is a phycobillin protein.  
     
     
         45 . A method for determining the presence or concentration of a target nucleic acid molecule in a sample which comprises: 
 (I) contacting said sample with an oligonucleotide-protein conjugate, wherein a sequence of an oligonucleotide portion of said conjugate is selected to be able to hybridize with said target nucleic acid molecule, wherein said oligonucleotide-protein conjugate is produced through the process comprising conjugating an oligonucleotide-heterofunctional linker conjugate to a protein having a thiolated group formed by thiolation of an amino group of said protein; 
 wherein:  
 said linker is conjugated to said oligonucleotide by a chemical bond formed between an atom of a first group of said linker and an amino group of said oligonucleotide, and wherein said linker possesses a second group that is reactive with said thiolated group of said protein and forms a chemical bond therewith; and  
   (II) detecting a protein portion of any of said oligonucleotide-protein conjugate having an oligonucleotide portion hybridized to said target nucleic acid molecule; wherein said detection determines the presence or concentration of said target nucleic acid molecule in said sample.    
     
     
         46 . The method of  claim 45 , wherein said amino group of said oligonucleotide is at the 3′ end of said oligonucleotide.  
     
     
         47 . The method of  claim 45 , wherein said amino group of said oligonucleotide is at the 5′ end of said oligonucleotide.  
     
     
         48 . The method of  claim 45 , wherein said amino group of said oligonucleotide is at an internal site of said oligonucleotide.  
     
     
         49 . The method of  claim 45 , wherein said protein of said oligonucleotide-protein is an enzyme, hapten, immunoglobulin, streptavidin, avidin, or a phycobillin protein.  
     
     
         50 . The method of  claim 49 , wherein said protein is an enzyme.  
     
     
         51 . The method of  claim 50 , wherein said enzyme is selected from the group consisting of alkaline phosphatase, β-galactosidase, horse radish peroxidase, and urease.  
     
     
         52 . The method of  claim 49 , wherein said protein is a hapten.  
     
     
         53 . The method of  claim 49 , wherein said protein is an immunoglobulin.  
     
     
         54 . The method of  claim 53 , wherein said immunoglobulin is an immunoglobulin that is able to bind to a drug, a receptor, a receptor ligand, or a tumor antigen.  
     
     
         55 . The method of  claim 53 , wherein said immunoglobulin is an immunoglobulin that is able to an antigen that is characteristic of a pathogen.  
     
     
         56 . The method of  claim 55 , wherein said pathogen is a virus.  
     
     
         57 . The method of  claim 55 , wherein said pathogen is a bacteria or fungus.  
     
     
         58 . The method of  claim 45 , wherein said target nucleic acid molecule is a nucleic acid molecule of a pathogen.  
     
     
         59 . The method of  claim 45 , wherein said target nucleic acid molecule is a nucleic acid molecule of a tumor cell.  
     
     
         60 . The method of  claim 49 , wherein said protein is a streptavidin protein.  
     
     
         61 . The method of  claim 49 , wherein said protein is an avidin protein.  
     
     
         62 . The method of  claim 49 , wherein said protein is a phycobillin protein.  
     
     
         63 . A method for determining the presence or concentration of a target analyte in a sample which comprises: 
 (I) contacting said sample with an oligonucleotide-protein conjugate, wherein a protein portion of said conjugate is selected to be able to bind to said target analyte, wherein said oligonucleotide-protein conjugate is produced through the process comprising conjugating an oligonucleotide-heterofunctional linker conjugate to a protein having a thiolated group formed by thiolation of an amino group of said protein; 
 wherein:  
 said linker is conjugated to said oligonucleotide by a chemical bond formed between an atom of a first group of said linker and an amino group of said oligonucleotide, and wherein said linker possesses a second group that is reactive with said thiolated group of said protein and forms a chemical bond therewith; and  
   (II) detecting an oligonucleotide portion of any of said oligonucleotide-protein conjugate having a protein portion bound to said target analyte; wherein said detection determines the presence or concentration of said target analyte in said sample.    
     
     
         64 . The method of  claim 63 , wherein said amino group of said oligonucleotide is at the 3′ end of said oligonucleotide.  
     
     
         65 . The method of  claim 63 , wherein said amino group of said oligonucleotide is at the 5′ end of said oligonucleotide.  
     
     
         66 . The method of  claim 63 , wherein said amino group of said oligonucleotide is at an internal site of said oligonucleotide.  
     
     
         67 . The method of  claim 63 , wherein said protein of said oligonucleotide-protein is an enzyme, receptor or receptor ligand.  
     
     
         68 . The method of  claim 67 , wherein said protein is an enzyme.  
     
     
         69 . The method of  claim 67 , wherein said protein is a receptor.  
     
     
         70 . The method of  claim 67 , wherein said protein is a receptor ligand.  
     
     
         71 . The method of  claim 67 , wherein said protein is a tumor antigen.  
     
     
         72 . The method of  claim 53 , wherein said protein is characteristic of a pathogen.  
     
     
         73 . The method of  claim 72 , wherein said pathogen is a virus.  
     
     
         74 . The method of  claim 72 , wherein said pathogen is a bacteria or fungus.