Nucleic acid hairpin probes and uses thereof

Abstract

This invention relates generally to nucleic acid hybridization analysis. More specifically, oligonucleotide probes containing hairpin structures, or arrays of such oligonucleotide probes immobilized on a solid support, that are suitable for hybridization analysis are provided. Methods for nucleic acid hybridization analysis using the probes or array of immobilized probes are also provided.

What is claimed is:  
     
         1 . An oligonucleotide probe for hybridization analysis, comprising a nucleotide sequence that forms a hairpin structure having a double stranded segment, under suitable conditions, wherein the double stranded segment is formed between two perfectly matched nucleotide sequences, and wherein at least a portion of at least one of the nucleotide sequences located within the double stranded segment is complementary to a target nucleotide sequence to be detected.  
     
     
         2 . The probe of  claim 1 , which comprises DNA or a derivative thereof.  
     
     
         3 . The probe of  claim 1 , which comprises RNA or a derivative thereof.  
     
     
         4 . The probe of  claim 1 , which comprises both DNA and RNA or derivatives thereof.  
     
     
         5 . The probe of  claim 1 , wherein the nucleotide sequence complementary to the target nucleotide sequence to be detected is completely located within the double stranded segment.  
     
     
         6 . The probe of  claim 1 , further comprising an element or a modification that facilitates intramolecular crosslinking of the probe upon suitable treatment.  
     
     
         7 . The probe of  claim 6 , wherein the element is a chemically or photoactively activatable crosslinking agent.  
     
     
         8 . The probe of  claim 7 , wherein the photoactively activatable crosslinking agent is a furocoumarin.  
     
     
         9 . The probe of  claim 6 , wherein the element is a macromolecule having multiple ligand binding sites.  
     
     
         10 . The probe of  claim 9 , wherein the macromolecule is a component of biotin-avidin binding system.  
     
     
         11 . The probe of  claim 1 , further comprising an element or a modification that renders the probe sensitive or resistant to nuclease digestion.  
     
     
         12 . The probe of  claim 11 , wherein the element is a restriction enzyme cleavage site.  
     
     
         13 . The probe of  claim 11 , wherein at least a portion of the double stranded segment of the probe is a duplex between a DNA strand and a RNA strand, the DNA strand containing methylphosphonates, and wherein at least a portion of the RNA strand is complementary to the target nucleotide sequence to be detected.  
     
     
         14 . An array of oligonucleotide probes immobilized on a solid support for hybridization analysis, comprising a solid support suitable for use in nucleic acid hybridization having immobilized thereon a plurality of oligonucleotide probes, each of the probes comprising a nucleotide sequence that forms a hairpin structure having a double stranded segment under suitable conditions, wherein the double stranded segment is formed between two complementary nucleotide sequences, and wherein at least a portion of at least one of the nucleotide sequences located within the double stranded segment is complementary to a target nucleotide sequence to be detected.  
     
     
         15 . The array of  claim 14 , wherein the double stranded segment of the hairpin structure is formed between two perfectly matched nucleotide sequences.  
     
     
         16 . The array of  claim 14 , wherein the double stranded segment of the hairpin structure is formed between two imperfectly matched nucleotide sequences.  
     
     
         17 . The array of  claim 14 , wherein the plurality of probes comprise DNA or a derivative thereof.  
     
     
         18 . The array of  claim 14 , wherein the plurality of probes comprise RNA or a derivative thereof.  
     
     
         19 . The array of  claim 14 , wherein the plurality of probes comprise both DNA and RNA or derivatives thereof.  
     
     
         20 . The array of  claim 14 , wherein the nucleotide sequence complementary to the target nucleotide sequence to be detected is completely located within the double stranded segment.  
     
     
         21 . The array of  claim 14 , wherein the probes further comprise an element or modification that facilitates intramolecular crosslinking of the probes upon suitable treatment.  
     
     
         22 . The array of  claim 21 , wherein the element is a chemically or photoactively activatable crosslinking agent.  
     
     
         23 . The array of  claim 22 , wherein the photoactively activatable crosslinking agent is a furocoumarin.  
     
     
         24 . The array of  claim 21 , wherein the element is a macromolecule having multiple ligand binding sites.  
     
     
         25 . The array of  claim 24 , wherein the macromolecule is a component of biotin-avidin binding system.  
     
     
         26 . The array of  claim 14 , wherein the probes further comprise an element or modification that renders the probe sensitive or resistant to nuclease digestion.  
     
     
         27 . The array of  claim 26 , wherein the element is a restriction enzyme cleavage site.  
     
     
         28 . The array of  claim 26 , wherein at least a portion of the double stranded segment of the probes is a duplex between a DNA strand and a RNA strand, said DNA strand contains methylphosphonates and at least a portion of said RNA strand is complementary to a target nucleotide sequence to be detected.  
     
     
         29 . A method for detecting a target nucleotide sequence in a sample comprising the steps of: 
 a) providing an oligonucleotide probe comprising a nucleotide sequence that forms a hairpin structure having a double stranded segment under suitable conditions, wherein the double stranded segment is formed between two complementary nucleotide sequences, and wherein at least a portion of at least one of the nucleotide sequences located within the double stranded segment is complementary to the target nucleotide sequence to be detected;    b) contacting the probe provided in step a) with a sample containing or suspected of containing the target nucleotide sequence under conditions that favor intermolecular hybridization between the probe and the target nucleotide sequence over intramolecular hybridization of the probe itself; and    c) assessing the intermolecular hybrid formed in step b).    
     
     
         30 . The method of  claim 29 , wherein the nucleotide sequence complementary to the target nucleotide sequence to be detected is completely located within the double stranded segment.  
     
     
         31 . The method of  claim 29 , wherein the conditions that favor intermolecular hybridization between the probe and the target nucleotide sequence over intramolecular hybridization of the probe itself is achieved by controlling compositions of the probe and the target nucleotide sequence so that the Tm of the intermolecular hybrid is higher than the Tm of the intramolecular hybrid.  
     
     
         32 . The method of  claim 31 , wherein the Tm of the intermolecular hybrid is at least 2° C. higher than the Tm of the intramolecular hybrid.  
     
     
         33 . The method of  claim 31 , wherein the intermolecular hybrid is a RNA:RNA hybrid whereas the intramolecular hybrid is a RNA:DNA or a DNA:DNA hybrid.  
     
     
         34 . The method of  claim 31 , wherein the intermolecular hybrid is a RNA:DNA hybrid whereas the intramolecular hybrid is a DNA:DNA hybrid.  
     
     
         35 . The method of  claim 29 , wherein the target nucleotide sequence is detectably labeled and the formation of the intermolecular hybrid is assessed by detecting the label of the target nucleotide sequence in the intermolecular hybrid.  
     
     
         36 . The method of  claim 35 , wherein the label is selected from the group consisting of a chemical, an enzymatic, an radioactive, a fluorescent, a luminescent and a FRET label.  
     
     
         37 . The method of  claim 29 , wherein the formation of the intermolecular hybrid is assessed by addition of a detectably labeled secondary probe that specifically hybridizes with at least a portion of the intermolecular hybrid and the detection of a secondary intermolecular hybrid formed between the secondary probe and the original intermolecular hybrid indicates the presence of the target nucleotide sequence in the sample.  
     
     
         38 . The method of  claim 37 , further comprising a step of crosslinking the intermolecular hybrid and the intramolecular hybrid after the formation of the intermolecular hybrid but before the addition of the detectably labeled secondary probe.  
     
     
         39 . The method of  claim 38 , wherein the crosslinking step is effected via addition of a crosslinking agent subsequent to hybridization of the original probe with the target nucleotide sequence.  
     
     
         40 . The method of  claim 38 , wherein the original hairpin probe is synthesized with the crosslinking agent attached and crosslinking is achieved by addition of an appropriate agent or treatment.  
     
     
         41 . The method of  claim 37 , wherein the secondary probe specifically hybridizes with a portion of the target nucleotide sequence that is not involved in the hybridization of the target nucleotide sequence and the original probe.  
     
     
         42 . The method of  claim 37 , wherein the secondary probe specifically hybridizes with a portion of the target nucleotide sequence that is involved in the hybridization of the target nucleotide sequence and the original probe and the nucleotide sequence in the original probe that is complementary to the same portion of the target nucleotide sequence is removed prior to or currently with the addition of the secondary probe.  
     
     
         43 . The method of  claim 42 , wherein the hairpin structure in the original probe is formed between a DNA strand that contains methylphosphonates and a RNA strand that is complementary to the target nucleotide sequence and wherein the RNA strand, after forming a hybrid with the target nucleotide sequence but before the addition of the secondary probe, is removed by a RNase H treatment.  
     
     
         44 . The method of  claim 37 , wherein the secondary probe specifically hybridizes with a portion of the original probe that is involved in the formation of the intramolecular hybrid but is not involved in the formation of the intermolecular hybrid with the target nucleotide sequence and wherein the same portion of the original probe that remains within the unhybridized original probe is removed prior to or currently with the addition of the secondary probe.  
     
     
         45 . The method of  claim 44 , wherein the portion of the original probe that remains within the unhybridized original probe contains a restriction enzyme cleavage site and is removed by cleavage with said restriction enzyme.  
     
     
         46 . The method of  claim 37 , wherein the detectably labeled secondary probe is a specific probe.  
     
     
         47 . The method of  claim 37 , wherein the detectably labeled probe is a degenerate probe.  
     
     
         48 . The method of  claim 29 , wherein the probe is immobilized on a solid support.  
     
     
         49 . The method of  claim 29 , wherein a plurality of the probes immobilized on a solid support is used.  
     
     
         50 . The method of  claim 29 , wherein the sample is a biosample.  
     
     
         51 . The method of  claim 29 , wherein a plurality of samples is assayed simultaneously.  
     
     
         52 . A method for detecting a target nucleotide sequence in a sample comprising the steps of: 
 a) providing an array of oligonucleotide probes immobilized on a solid support suitable for use in nucleic acid hybridization having immobilized thereon a plurality of oligonucleotide probes, each of the probe comprising a nucleotide sequence that forms a hairpin structure having a double stranded setment under suitable conditions, wherein the double stranded segment is formed between two complementary nucleotide sequences, and wherein at least a portion of at least one of the nucleotide sequences located within the double stranded segment is complementary to a target nucleotide sequence to be detected;    b) contacting the array of probes provided in step a) with a sample containing or suspected of containing the target nucleotide sequence under conditions that favor intermolecular hybridization between the probes and the target nucleotide sequence over intramolecular hybridization of the probes themselves; and    c) assessing the intermolecular hybrids formed in step b).    
     
     
         53 . The method of  claim 52 , wherein the double stranded segment of the hairpin structure is formed between two perfectly matched nucleotide sequences.  
     
     
         54 . The method of  claim 52 , wherein the double stranded segment of the hairpin structure is formed between two imperfectly matched nucleotide sequences.  
     
     
         55 . The method of  claim 52 , wherein a plurality of samples is assayed simultaneously.  
     
     
         56 . The method of  claim 37 , wherein the detectably labeled secondary probe is crosslinked to the intermolecular hybrid before the detection of the secondary intermolecular hybrid formed between the secondary probe and the original intermolecular hybrid.