Polynucleotide detection method employing self-reporting dual inversion probes

Abstract

Method of detecting a target polynucleotide based on the use of dual inversion hybridization probes having stem-and-loop structures, wherein the stem portion of the structure comprises a pair of interactive arms that are substantially prevented from interacting with target polynucleotides. The arms of the dual inversion hybridization probes interact in a conventional antiparallel fashion, but have backbone polarities opposite that of the target-complementary loop portion of the probe. Arm portions of the dual inversion probes do not substantially contribute to sequence-dependent stabilization of probe:target hybrids. Incorporating inversion linkages into the structures of these probes dramatically simplifies the process of designing stem-and-loop hybridization probes.

1 . A method of detecting a target polynucleotide in a test sample, comprising the steps of: 
 providing a dual inversion hybridization probe;    contacting said dual inversion hybridization probe with any of said target polynucleotide that may be present in the test sample under hybridization-promoting conditions; and    detecting the formation of hybrid duplexes comprising said dual inversion hybridization probe and said target polynucleotide as an indication of the presence of said target polynucleotide sequence in said test sample, 
 wherein said dual inversion hybridization probe comprises 
 (a) a loop comprising a target-complementary sequence of bases joined to a loop backbone, said target-complementary sequence of bases extending from a first boundary thereof to a second boundary thereof,  
 (b) a first arm joined to said target-complementary sequence of bases at said first boundary thereof through a first arm linkage, said first arm comprising a first arm sequence of bases joined to a first arm backbone,  
 (c) a second arm joined to said target-complementary sequence of bases at said second boundary thereof through a second arm linkage, said second arm comprising a second arm sequence of bases joined to a second arm backbone, 
 wherein both said first arm linkage and said second arm linkage are inversion linkages different from each other, said inversion linkages optionally including a non-nucleotide linker, and  
 
 (d) at least one detectable label joined to any of said loop, said first arm, said second arm or, if present, said non-nucleotide linker, 
 wherein said first arm and said second arm interact with each other in the absence of said target polynucleotide to form a stem duplex.  
 
 
   
     
     
         2 . The method of  claim 1 , wherein said at least one detectable label of said dual inversion hybridization probe comprises a pair of interactive labels comprising a first label and a second label, said first label being joined to said first arm and said second label being joined to said second arm.  
     
     
         3 . The method of  claim 1 , wherein said first arm linkage of said dual inversion hybridization probe is a 3′-3′ inversion linkage, and wherein said second arm linkage of said dual inversion hybridization probe is a 5′-5′ inversion linkage.  
     
     
         4 . The method of  claim 1 , wherein said first arm linkage of said dual inversion hybridization probe is a 5′-5′ inversion linkage, and wherein said second arm linkage of said dual inversion hybridization probe is a 3′-3′ inversion linkage.  
     
     
         5 . The method of  claim 2 , wherein at least one of said loop, said first arm or said second arm of said dual inversion hybridization probe comprises at least one nucleotide analog.  
     
     
         6 . The method of  claim 5 , wherein said loop of said dual inversion hybridization probe comprises 2′-methoxy nucleotide analogs.  
     
     
         7 . The method of  claim 2 , wherein the target-complementary sequence of bases of said loop of said dual inversion hybridization probe has a length in the range of from 10-25 bases.  
     
     
         8 . The method of  claim 7 , wherein the target-complementary sequence of bases of said loop of said dual inversion hybridization probe has a length in the range of from 16-22 bases.  
     
     
         9 . The method of  claim 7 , wherein the first arm of said dual inversion hybridization probe has a length of from 5-12 bases.  
     
     
         10 . The method of  claim 9 , wherein the second arm of said dual inversion hybridization probe has a length of from 5-12 bases.  
     
     
         11 . The method of  claim 7 , wherein both the first arm and the second arm of said dual inversion hybridization probe have lengths in the range of from 6-8 bases.  
     
     
         12 . The method of  claim 2 , wherein said pair of interactive labels of said dual inversion hybridization probe is a pair of FRET interactive labels.  
     
     
         13 . The method of  claim 2 , wherein said pair of interactive labels of said dual inversion hybridization probe is a pair of non-FRET interactive labels.  
     
     
         14 . The method of  claim 13 , wherein one member of said pair of non-FRET interactive labels of said dual inversion hybridization probe comprises fluorescein.  
     
     
         15 . The method of  claim 10 , wherein said pair of interactive labels of said dual inversion hybridization probe is a pair of FRET interactive labels.  
     
     
         16 . The method of  claim 10 , wherein said pair of interactive labels of said dual inversion hybridization probe is a pair of non-FRET interactive labels.  
     
     
         17 . The method of  claim 1 , wherein the detecting step comprises detecting by fluorometry.