Diastereomeric linking reagents for nucleotide probes

Abstract

A versatile reagent with a non-nucleotide monomeric unit comprising an enantiomeric center and having a ligand, and first and second coupling groups that are linked to the non-nucleotide monomeric unit. Such a reagent permits preparation of versatile nucleotide/non-nucleotide polymers, having any desired sequence of nucleotide and non-nucleotide monomeric units, each of the latter of which bear a desired ligand. These polymers can, for example, be used as probes which can exhibit enhanced sensitivity and/or which are capable of detecting a genus of nucleotides each species of which has a common target nucleotide sequence of interest bridged by different sequences not of interest.

1 . A diastereomeric linking reagent comprising a phosphoramidite based compound having the formula:
   D-X 1 —R 2 (X 3 -M) n -Z or Z—X 1 —R 2 (X 3 -M) n -D
   wherein:
 Z is either a reactive phosphorus-containing group attached to the end of R 2 , an OH group of a first nucleotide or a DNA synthesis support attached to the end of R 2  through a cleavable ester linkage; 
 R 2  comprises a chain of atoms that is 2-20 atoms in length joined to X 1  and Z, wherein R 2  is stable to DNA synthesis and deprotection conditions; 
 X 1  is either O, S, NH or —N═N—; 
 D is an alterable or removable protecting group which, upon alteration or removal permits X 1  to be coupled to the phosphorus group of a second nucleotide; 
 each X 3  is independently a linker-arm joined at a first end to R 2  and joined at a second end to M, wherein each X 3  is stable to DNA synthesis and deprotection conditions; 
 each M is independently either a label stable to DNA synthesis and deprotection conditions or an alterable or removable protecting group which, upon alteration or removal, permits X 3  to be coupled to a label; and 
 n is a positive integer. 
   
     
     
         2 . A diastereomeric linking reagent comprising a phosphoramidite based compound having the formula: 
       
         
           
           
               
               
           
         
         wherein:
 Z is either a reactive phosphorus-containing group, an OH group of a first nucleotide or a cleavable ester attached to a DNA synthesis support; 
 D is an alterable or removable protecting group which, upon alteration or removal permits said non-nucleotide linking reagent to be coupled to a second nucleotide; 
 i is either 0, 1, 2 or 3, and j is either 0, 1, 2 or 3, provided that i+j is at least 1; 
 L 1  is a first linker-arm; 
 L 2  is either H or a non-linking alkyl chain or a non-linking mixed alkyl chain or a second linker-arm; 
 
         wherein said first linker-arm, and said second linker-arm each independently have the formula: (CH 2 ) k —NH—(CO—(CH 2 ) q NH) r -M; 
         wherein each M is independently H, fluorenylmethoxycarbonyl, trifluoroacetyl or a label stable to DNA synthesis and deprotection conditions, 
         each k is independently an integer between 0 and 4, inclusive, 
         each q is independently an integer between 1 and 11, inclusive, 
         each r is independently either 0, 1, or 2, provided that each k+1+(2+q)r in said first linker-arm, and in said second linker-arm are independently an integer between 1 and 25, inclusive, and 
         wherein the compound is an R enantiomer. 
       
     
     
         3 . The linking reagent of  claim 1 , wherein the compound is an R enantiomer. 
     
     
         4 - 56 . (canceled)