Nucleic acid probes having highly hydrophilic non-nucleosidic tags with multiple labels, and uses thereof

Abstract

Highly hydrophilic non-nucleosidic tags with multiple labels are provided for use in nucleic acid probes. The tags are branched structures having a phosphodiester backbone, which have the advantages of a small dimensional size and high hydrophilicity. After the tag is labeled, its high negative charge and minimal size help to keep the carriers away from DNA or RNA molecules, due to repulsion between negative charges. Non-specific intercalation and steric hindrance are therefore minimized, and the hydrophobicity, if any of reporter molecules is reduced. The probes are used in place of conventionally labeled oligonucleotides for a variety of hybridization reactions.

We claim:  
     
         1 . A method of synthesizing a probe comprising a nucleic acid moiety and at least one tag moiety, wherein said tag moiety has the structure:  
       
         
           
           
               
               
           
         
       
       where Lp is a spacing monomer, Np is an internal monomer providing an attachment site for a label, tNp is an internal monomer or a monovalent 5′ amino-modifier, and Yp is a bi-branching monomer having the structure:  
       
         
           
           
               
               
           
         
       
       wherein R 1  and R 2  are independently chosen from hydrogen and lower alkyl, R 3  is β-cyanoethyl or methyl, and R 4  is a protecting group for a primary hydroxyl group, and wherein, independently, n is from 0 to 20, n 1  is from 0 to 20, n 2  is from about 0 to 20, x is from 0 to 9, and y is from 0 to 9, wherein said method comprises the steps of: 
 synthesizing said nucleic acid by stepwise addition of reactive nucleotide phosphoramidite monomers; and  
 adding to said nucleic acid non-nucleosidic phosphoramidite monomers to provide the tag moiety.  
 
     
     
         2 . The method of  claim 1 , further comprising adding multiple tag moieties.  
     
     
         3 . The method of  claim 1 , wherein Lp is provided by addition of the monomer:  
       
         
           
           
               
               
           
         
       
       wherein R 1  and R 2  are independently chosen from hydrogen and lower alkyl, R 3  is β-cyanoethyl or methyl, and R 4  is a protecting group for a primary hydroxyl group.  
     
     
         4 . The method of  claim 1 , wherein both Np and tNp are provided by addition of a phosphoramidite compound comprising one or more amino groups.  
     
     
         5 . The method of  claim 4 , further comprising the steps of: attaching one or more label(s) to said amino group(s).  
     
     
         6 . The method of  claim 5 , wherein said label is a detectable compound.  
     
     
         7 . The method of  claim 5 , wherein said label is a binding compound.  
     
     
         8 . The method of  claim 5 , wherein said label provides a specific cleavage site.  
     
     
         9 . The method of  claim 5 , wherein said nucleic acid is a synthetic single-stranded oligonucleotide with or without branched segment, that is capable of hybridizing to a single-stranded nucleic acid of interest.  
     
     
         10 . A method for detection of a target nucleic acid, the method comprising: 
 combining a sample suspected of comprising said target nucleic acid with a probe comprising a nucleic acid moiety and at least one tag moiety, wherein said tag moiety has the structure:                          where Lp is a spacing monomer, Np is an internal monomer providing an attachment site for a label, tNp is an internal monomer at a terminal position providing an attachment site for a label or a monovalent 5′ amino-modifier, and Yp is a bi-branching monomer having the structure:                          wherein the sample and probe are combined under conditions wherein said probe specifically hybridizes to said target nucleic acid;    washing away unhybridized probe; and    detecting the presence of said probe.    
     
     
         11 . The method of  claim 10 , comprising two or more tag moieties.  
     
     
         12 . The method of  claim 10 , wherein both Np and tNp provide an amino group.  
     
     
         13 . The method of  claim 10 , wherein Lp has the structure:  
       
         
           
           
               
               
           
         
       
       where n is 0 or 1.  
     
     
         14 . The method of  claim 12 , further comprising one or more label(s) attached to said amino group(s).  
     
     
         15 . The method of  claim 14 , wherein said label is a detectable compound.  
     
     
         16 . The method of  claim 15 , wherein said label is a binding compound, and said method further comprises the steps of: 
 combining said sample with a reagent that specifically binds to said label; and    detecting the presence of said reagent.    
     
     
         17 . The method of  claim 10 , wherein nucleic acids in said sample are bound to a solid support.  
     
     
         18 . The method of  claim 10 , wherein said nucleic acid is a synthetic single-stranded oligonucleotide with or without branched segment, that is capable of hybridizing to a single-stranded nucleic acid of interest.  
     
     
         19 . A method for detection of a target nucleic acid, the method comprising: 
 combining a sample suspected of containing said target nucleic acid, wherein nucleic acids in said sample are attached to a solid support via a cleavage site, with a probe comprising a nucleic acid moiety and at least one tag moiety, wherein said tag moiety has the structure:                          where Lp is a spacing monomer, Np is an internal monomer providing an attachment site for a label, tNp is an internal monomer at a terminal position providing an attachment site for a label or a monovalent 5′ amino-modifier, and Yp is a bi-branching monomer having the structure:                          wherein the sample and probe are combined under conditions wherein said probe specifically hybridizes to said target nucleic acid;    washing away unhybridized probe;    cleaving said probe at said specific cleavage sites to release said target DNA;    moving said released target DNA to a separate container; and    detecting the presence of said target DNA.