Sets of generalized target-binding e-tag probes

Abstract

Probe sets for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided. Detection involves the release of identifying tags as a consequence of target recognition. The probe sets include electrophoretic tag probes or e-tag probes, comprising a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. Target antiligands are contacted with a set of e-tag probes and the contacted antiligands are treated with a selected cleaving agent resulting in a mixture of e-tag reporters and uncleaved and/or partially cleaved e-tag probes. The mixture is exposed to a capture agent effective to bind to uncleaved or partially cleaved e-tag probes, followed by electrophoretic separation. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification.

It is claimed:  
     
         1 . A set of electrophoretic tag (e-tag) probes for detecting the binding of or interaction between each or any of a plurality of ligands and one or more target antiligands, the set comprising j members, and each of said e-tag probes having the form:  
       (D, M j )-L-T j , where  (a) D is a detection group comprising a detectable label;    (b) T j  is a ligand capable of binding to or interacting with a target antiligand,    (c) L is a linking group connected to T j  by a bond that is cleavable by a selected cleaving agent when the probe is bound to or interacting with the target antiligand, wherein cleavage by said agent produces an e-tag reporter of the form (D, M j )-L′, where L′ is the residue of L attached to (D, M j ) after such cleavage,    (d) M j  is a mobility modifier having a charge/mass ratio that imparts a unique and known electrophoretic mobility to a corresponding e-tag reporter of the form (D, M j )-L′, within a selected range of electrophoretic mobilities with respect to other e-tag reporters of the same form in the probe set; and    (e) (D, M j )-includes both D-M j - and M j -D-;    said uncleaved or partially cleaved e-tag probes, but not the corresponding e-tag reporter, having one or more chemical groups capable of reacting with or binding to a selected capture agent that is effective to 
 (i) impart a mobility to the probes bound to capture agent that prevents the probes from electrophoretically migrating within said range of electrophoretic mobilities or  
 (ii) immobilize the probes on a solid support.  
   
     
     
         2 . The probe set of  claim 1 , for detecting each or any of a plurality of known, selected target nucleotide sequences, the set comprising j members, wherein: 
 (a) T j  is an oligonucleotide target-binding moiety having a sequence of nucleotides U i  connected by intersubunit linkages B i, i+1 , where i includes all integers from 1 to n, and n is sufficient to allow the target-binding moiety to hybridize specifically with a target nucleotide sequence;    (b) L is a nucleotide joined to U i  in T j  through a nuclease-cleavable bond; and    (c) each of the target-binding moieties contains at least one modification selected from the following: 
 (i) at least one nuclease-resistant bond B 1, i+1 , where i includes at least 1;  
 (ii) U i  containing a capture ligand capable of binding specifically to a capture agent; and  
 (iii) a nuclease-resistant bond B i, i+1 , where i includes at least 1, and at least one nucleotide U i  containing a capture ligand capable of binding specifically to a capture agent, where i≧1.  
   
     
     
         3 . The probe set of  claim 1 , wherein L includes at least a portion of an amino acid sequence that is recognized and cleaved by a selected peptidase.  
     
     
         4 . The probe set of  claim 1 , wherein L includes at least a portion of an oligosaccharide that is recognized and cleaved by a selected hydrolytic enzyme.  
     
     
         5 . The probe set of  claim 1 , wherein L and T j  are linked by an ester linkage that is cleaved by a selected esterase.  
     
     
         6 . The probe set of  claim 1 , wherein L and T j  are linked by a disulfide bond, and the antiligand is attached to an oxidase enzyme, such that in the presence of a substrate for the enzyme, H 2 O 2  generated by the oxidase is effective to cleave the disulfide linkage in a probe bound to the antiligand.  
     
     
         7 . The probe of  claim 1 , wherein L and T j  are linked by a bond cleavable by singlet oxygen, wherein the antiligand is attached to a sensitizer capable of generating singlet oxygen when photoactivated.  
     
     
         8 . The probe set of  claim 1 , for use in detecting the binding of each or any of a plurality of ligands to a target antiligand molecule, wherein the plurality of ligands are represented by T j . 
 How is this limiting to  claim 1 ?   
     
     
         9 . The probe set of  claim 1 , for use in screening for a ligand capable of binding to a receptor, wherein the ligands are represented by T j  and are members of a combinatorial library of small organic molecules, and the antiligand is the receptor. 
 NOTE: This claim was originally written as a dependent claim to  claim 7  above.    
     
     
         10 . The probe set of  claim 1 , for use in screening substrates of a selected enzyme antiligand, wherein the substrate comprises a fixed moiety L and a variable moiety T j , and interaction of a substrate probe with the enzyme is effective to cleave the substrate to release the T j  moiety from the substrate. 
 NOTE: This claim was originally written as a dependent claim to  claim 7  above.    
     
     
         11 . The probe set of  claim 1 , wherein each M j  has a unique charge/mass ratio by virtue of variations in mass, but not charge.  
     
     
         12 . The probe set of  claim 1 , wherein each M j  has a unique charge/mass ratio, by virtue of changes in both mass and charge.  
     
     
         13 . The probe set of  claim 12 , containing at least 5 probes whose corresponding e-tag reporters have unique charge/mass ratios of between −0.001 and 0.5. 
 NOTE: The 033.00 US spec (page 10, line 23) cited a “range of about −0.0001 to 0.1, usually in the range of about −0.001 to about 0.5.” The 0.1 was thought to be in error.  
 
     
     
         14 . The probe set of  claim 12 , containing at least 9 probes whose corresponding e-tag reporters have unique charge/mass ratios of between −0.001 and 0.5.  
     
     
         15 . The probe set  claim 12 , wherein each M j  is formed of a selected number of negatively charged and/or positively charged amino acids.  
     
     
         16 . The probe set of  claim 12 , wherein each M j  includes an alkyl chain, and differs from other M j  in the set by 1-3 methylene groups in the chain.  
     
     
         17 . The probe set  claim 1 , wherein the detectable label is selected from the group consisting of a fluorophore, a chromophore, and an electrochemical compound capable of a detectable reaction in the presence of a redox agent.  
     
     
         18 . The probe set of  claim 1 , wherein the detectable label has a selected mass and charge.  
     
     
         19 . The probe set of  claim 18 , containing subsets of probes, each subset having a label with a unique mass/charge ratio.  
     
     
         20 . The probe set of claims  18  and  19 , wherein the detectable label is a fluorophore.