Multipartite high-affinity nucleic acid probes

Abstract

The invention provides a collection of probes useful for hybridizing to a target nucleic acid. The probes associate with each other, binding with high affinity to the target nucleic acid, to form three-way junctions and other complexes. At least one of the probes in each collection includes a nucleic acid analog. Methods using the probes in hybridization and as primers are also provided.

What is claimed is:  
     
         1 . A collection of probes for use in hybridizing to a target nucleic acid, the collection comprising a first probe and a second probe, 
 the first probe comprising a first portion complementary to a first region of a target nucleic acid and capable of hybridizing thereto, and a second portion capable of hybridizing with the second probe, wherein the first and second portions of the first probe are joined by a flexible linker; and    the second probe comprising a first portion complementary to a second region of the target nucleic acid and capable of hybridizing thereto, and a second portion capable of hybridizing with the first probe;    wherein each of the first and the second region of the target nucleic acid is from three to eight nucleotides in length; and    wherein at least one of the first probe or the second probe comprises a high-affinity nucleic acid analog.    
     
     
         2 . The collection of  claim 1  wherein the second portion of the first probe and the second portion of the second probe comprise regions which are complementary to each other; and 
 wherein the first and second portions of the second probe are joined by a flexible linker.  
 
     
     
         3 . The collection of  claim 1  wherein the second portion of the first probe hybridizes with a greater binding affinity with the second portion of the second probe than with any portion of the target nucleic acid.  
     
     
         4 . The collection of  claim 1  wherein the portion of the first probe that may be complementary to the first region comprises at least a portion of the high-affinity nucleic acid analog.  
     
     
         5 . The collection of  claim 1  wherein the high-affinity nucleic acid analog comprises one or more PNA monomer units.  
     
     
         6 . The collection of  claim 5  wherein the PNA has a 2-aminoethylglycine backbone.  
     
     
         7 . The collection of  claim 1  wherein at least one of the first probe or the second probe comprises a detection moiety.  
     
     
         8 . The collection of  claim 7  wherein the detection moiety is a fluorescent moiety, and wherein at least the first probe or the second probe comprises a quenching moiety.  
     
     
         9 . The collection of  claim 1  wherein at least one of the first probe or the second probe comprises an oligonucleotide sequence.  
     
     
         10 . The collection of  claim 1  wherein the flexible linker is 1 to 6 ethyleneoxy units, alkyldiyl of 1 to 20 carbon atoms, or aryldiyl of 6 to 20 carbon atoms.  
     
     
         11 . The collection of  claim 1  wherein the hybridization between the first probe and the second probe is mediated at least in part by a triple helix.  
     
     
         12 . The collection of  claim 1  wherein the first region of the target nucleic acid and the second region of the target nucleic acid are substantially adjacent.  
     
     
         13 . The collection of  claim 1  wherein a probe is immobilized on a solid support.  
     
     
         14 . The collection of  claim 1  wherein the target nucleic acid is immobilized on a solid support.  
     
     
         15 . The collection of  claim 1  comprising an array of a plurality of first probes and an array of a plurality of second probes, wherein 
 the portion of each of the first probes which may be complementary to the first region of the target nucleic acid has a different sequence, wherein the first region of the target nucleic acid is x nucleotides in length;  
 wherein the array of first probes comprises at least (0.5×4 x )÷N first probes, and wherein N is equal to the degeneracy of each of the first portions of each of the first probes; and  
 the portion of each of the second probes which may be complementary to the second region of the target nucleic acid has a different sequence.  
 
     
     
         16 . The collection of  claim 15  wherein the target nucleic acid is immobilized on a solid support.  
     
     
         17 . The collection of  claim 15  wherein the second region of the target nucleic acid is y nucleotides in length, and wherein the array of second probes comprises at least (0.5×4 y )÷M second probes, wherein M is equal to the degeneracy of each of the first portions of each of the second probes.  
     
     
         18 . The collection of  claim 17  wherein (x+y) is from 6 to 12.  
     
     
         19 . The collection of  claim 1 , wherein neither the first probe nor the second probe comprises a destabilizing moiety.  
     
     
         20 . A method of determining sequence information of a target nucleic acid, comprising the steps of: 
 (a) providing the collection of probes of  claim 1;     (b) exposing the target nucleic acid to the first probe and second probe to form a complex with the target nucleic acid, if the first region and the second region of the target nucleic acid is present; and    (c) detecting the presence or absence of the complex.    
     
     
         21 . The method of  claim 20  wherein the second portion of the first probe hybridizes with the second portion of the second probe prior to the formation of the complex with the target nucleic acid.  
     
     
         22 . The method of  claim 20  wherein the step of detecting comprises fluorescent analysis.  
     
     
         23 . The method of  claim 20  wherein the presence of the complex indicates the presence of a single nucleotide polymorphism.  
     
     
         24 . The method of  claim 20  wherein the complex is formed only if the first region and the second region of the target nucleic acid are present and substantially adjacent.  
     
     
         25 . The method of  claim 20  wherein the target nucleic acid is immobilized on a solid support.  
     
     
         26 . A method of determining sequence information of a target nucleic acid comprising the steps of: 
 (a) providing a collection of probes of  claim 15;     (b) exposing the target nucleic acid to a first probe having a known portion which may be complementary to the first region of the target nucleic acid;    (c) exposing the target nucleic acid to a second probe having a known portion which may be complementary to the second region of the target nucleic acid;    (d) forming a complex comprising the first probe, the second probe, and the target nucleic acid, if the target sequence is present;    (e) detecting the presence or absence of the complex; and    (f) repeating steps (b) to (e), if necessary, using at least a different first probe or a different second probe.    
     
     
         27 . The method of  claim 26  wherein the second portion of the first probe hybridizes with the second portion of the second probe prior to the formation of the complex with the target nucleic acid.  
     
     
         28 . The method of  claim 26  wherein the target nucleic acid is immobilized on a solid support.  
     
     
         29 . A method of priming an enzyme-catalyzed reaction, the method comprising the steps of: 
 (a) providing a collection of probes of  claim 1;     (b) exposing the target nucleic acid to a first probe having a known portion which may be complementary to the first region of the target nucleic acid;    (c) exposing the target nucleic acid to a second probe having a known portion which may be complementary to the second region of the target nucleic acid;    (d) forming a complex comprising the first probe, the second probe, and the target nucleic acid, if the target sequence is present; and    (e) providing an enzyme, such that the complex primes an enzymatic reaction.    
     
     
         30 . The method of  claim 29 , wherein the enzyme is a DNA-dependent DNA polymerase.  
     
     
         31 . The method of  claim 29  wherein the target nucleic acid is immobilized on a solid support.  
     
     
         32 . A kit comprising the collection of  claim 1  and a buffer.  
     
     
         33 . The kit of  claim 32 , further comprising an enzyme.  
     
     
         34 . The kit of  claim 32 , further comprising a detection moiety.