Reduction of nonspecific hybridization by using novel base-pairing schemes

Abstract

Methods are provided for substantially reducing background signals encountered in nucleic acid hybridization assays. The method is premised on the elimination or significant reduction of the phenomenon of nonspecific hybridization, so as to provide a detectable signal which is produced only in the presence the target polynucleotide of interest. In addition, a novel method for the chemical synthesis of isoguanosine or 2′-deoxy-isoguanosine is provided. The invention also has applications in antisense and aptamer therapeutics and drug discovery.

We claim:  
     
         1 . In a nucleic acid hybridization assay for detecting a nucleic acid analyte in a sample using a plurality of assay components each of which comprises at least one hybridizing oligonucleotide segment, the improvement which comprises incorporating into at least one hybridizing oligonucleotide segment a first nucleotidic unit which will not effectively base pair with adenosine (A), thymidine (T), cytidine (C), guanosine (G) or uridine (U) under conditions in which A-T and G-C base pairs are formed.  
     
     
         2 . The method of    claim 1   , wherein the first nucleotidic unit is capable of forming a base pair with a second, complementary nucleotidic unit.  
     
     
         3 . The method of    claim 2   , wherein the first and second nucleotidic units are incorporated into hybridizing oligonucleotide segments of assay components which are not complementary to nucleic acid segments of the nucleic acid analyte.  
     
     
         4 . The method of    claim 3   , wherein the first and second nucleotidic units are interchangeably selected from the group of complementary base pairs consisting of: 
                   
       wherein R is a backbone which will allow the first and second nucleotidic units to form a base pair with a complementary nucleotidic unit when incorporated into a polynucleotide, and R′ is hydrogen, methyl, α- or β-propynyl, bromine, fluorine or iodine.  
     
     
         5 . The method of    claim 4   , wherein the first and second nucleotidic units have the structure (I) 
                   
     
     
         6 . The method of    claim 1   , wherein the nucleic acid hybridization assay is a solution phase sandwich hybridization assay comprising (a) binding the analyte directly or indirectly to a solid support, (b) labelling the analyte, and (c) detecting the presence of analyte-associated label,  
     
     
         7 . In a nucleic acid hybridization assay for detecting a nucleic acid analyte in a sample using a plurality of assay components each of which comprises at least one hybridizing oligonucleotide segment, 
 the improvement which comprises incorporating T m1  hybrid complexes and T m2  hybrid complexes such that assay stringency can be controlled to selectively destabilize the T m1  hybrid complexes.    
     
     
         8 . The assay of    claim 7   , wherein the T m1  hybrid complexes comprise at least one of the hybrid complexes selected from the group consisting of a capture probe/capture extender complex, a label extender/amplifier complex and a label extender/preamplifier complex, and wherein the label comprises a member of a T m2  hybrid complex.  
     
     
         9 . The assay of    claim 7   , wherein the T m2  hybrid complexes comprise one or more of the complexes selected from the group consisting of a label extender/amplifier complex, a label extender/preamplifier complex and a label/amplifier complex, with the proviso that the label comprises a member of a T m2  complex.  
     
     
         10 . The assay of    claim 7   , wherein the assay stringency is controlled by altering formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent content or temperature.  
     
     
         11 . The assay of    claim 10   , wherein the assay stringency is controlled by altering the pH.  
     
     
         12 . The assay of    claim 10   , wherein the assay stringency is controlled by altering the salt concentration.  
     
     
         13 . The assay of    claim 11   , further comprising inhibiting the detection of nonspecifically bound label.  
     
     
         14 . The assay of    claim 13   , wherein the step of inhibiting the detection of nonspecifically bound label is effected by coating the solid support with an opaque substance, a label inhibitor or a luminescence inhibitor, absorber or quencher, providing a layer of oil over the nonspecifically bound label, or transferring the target-associated label to another vessel for label detection.  
     
     
         15 . The assay of    claim 6   , which further comprises incorporating T m1  hybrid complexes and T m2  hybrid complexes such that assay stringency can be controlled to selectively destabilize the T m1  hybrid complexes.  
     
     
         16 . In a solution phase sandwich hybridization assay for detecting a nucleic acid analyte in a sample using a plurality of assay components each of which comprises at least one hybridizing oligonucleotide segment, comprising (a) binding the analyte directly or indirectly to a solid support, (b) labelling the analyte, and (c) detecting the presence of analyte-associated label, 
 the improvement which comprises incorporating into at least one hybridizing oligonucleotide segment a first nucleotidic unit a first nucleotidic unit which will not effectively base pair with adenosine (A), thymidine (T), cytidine (C), guanosine (G) or uridine (U) under conditions in which A-T and G-C base pairs are formed.    
     
     
         17 . In a solution phase sandwich hybridization assay for detecting a nucleic acid analyte in a sample using a plurality of assay components each of which comprises at least one hybridizing oligonucleotide segment, comprising (a) binding the analyte directly or indirectly to a solid support, (b) labelling the analyte, and (c) detecting the presence of analyte-associated label, 
 the improvement which comprises incorporating T m1  hybrid complexes and T m2  hybrid complexes such that assay stringency can be controlled to selectively destabilize the T m1  hybrid complexes.    
     
     
         18 . A method for synthesizing a compound having the structural formula 
                   
       wherein R′ is selected from the group consisting of hydrogen, hydroxyl, sulfhydryl, halogeno, amino, alkyl, allyl and -OR 2 , where R 2 is alkyl, allyl, silyl or phosphate, comprising: 
 a) reacting a compound having the structural formula 
                 
 
 with a reagent suitable to protect both the 3′ and 5′ hydroxyl groups;  
 b) reacting the product of step (a) with a reagent suitable to convert the O 6 -oxy moiety into a functional group which is susceptible to nucleophilic displacement, thereby producing a functionalized O 6  moiety;  
 c) oxidizing the 2-amino group of the product of step (b);  
 d) reacting the product of step (c) with a nucleophilic reagent to displace the functionalized O 6  moiety; and  
 e) reacting the product of step (d) with a reagent suitable to deprotect the protected 3′ and 5′ hydroxyl groups.  
 
     
     
         19 . A method for synthesizing 2′-deoxy-iso-guanosine comprising: 
 a) converting 2′-deoxyguanosine to 3′, 5′-O-(t-butyldimethylsilyl) 2 -2′-deoxyguanosine by reacting 2′-deoxyguanosine with t-butyldimethylsilyl (TBDMS) chloride;  
 b) converting 3′, 5′-O-TBDMS 2 -2′-deoxyguanosine to O 6  (4-toluenesulfonyl)-3′,5′-O-TBDMS 2 -2′-deoxyguanosine by reacting 3′,5′-O-TBDMS 2 -2′-deoxyguanosine with 4-toluenesulfonyl chloride;  
 c) displacing the O 6 -(4-toluenesulfonyl) group by treating O 6 -(4-toluenesulfonyl)-3′,5′-O-TBDMS 2 -2′-deoxyguanosine with a phenol to give O 6 -(4-(methylthio)phenyl)-3′, 5′-O-TBDMS 2 -2′-deoxyguanosine;  
 d) oxidizing the 2-amino group of O 6 -(4-(methylthio)phenyl)-3′, 5′-O-TBDMS 2 -2′-deoxyguanosine to the oxy function by treating O 6 -(4-(methylthio)phenyl)-3′,5′-O-TBDMS 2 -2′-deoxyguanosine with t-butyl nitrite under neutral conditions to give O 6 -(4-(methylthio)-phenyl)-3′, 5′-O-TBDMS 2 -2′-deoxyxantosine; and  
 e) displacing the O 2 -(4-(methylthio)phenyl) group of O 6 -(4-(methylthio)phenyl)-3′,5′-O-TBDMS 2 -2′-deoxyxantosine with ammonium hydroxide at elevated temperature to give 3′, 5′-O-TBDMS 2 -2′-deoxy-isoguanosine.  
 
     
     
         20 . A kit for detecting a nucleic acid analyte in a sample, comprising at least one hybridizing oligonucleotide probe, a segment of which is capable of forming a hybrid complex with the analyte, and a means for detecting the hybrid complex, wherein the at least one hybridizing oligonucleotide probe comprises a first nucleotidic unit which will not effectively base pair with adenosine (A), thymidine (T), cytidine (C), guanosine (G) or uridine (U) under conditions in which A-T and G-C base pairs are formed.  
     
     
         21 . The kit of    claim 20    comprising: 
 (a) a set of capture probes, wherein said capture probes comprise a first nucleotidic unit which will not effectively base pair with A, T, C, G or U under conditions in which A-T and G-C base pairs are formed;  
 (b) a set of capture extender molecules comprising first and second hybridizing oligonucleotide segments, wherein the first hybridizing oligonucleotide segment is capable of forming hybrid complexes with the capture probes and the second hybridizing oligonucleotide segment is capable of forming hybrid complexes with predetermined segments of the nucleic acid analyte;  
 (c) label extender molecules comprising third and fourth hybridizing oligonucleotide segments, wherein the third hybridizing oligonucleotide segment is capable of forming hybrid complexes with segments of the nucleic acid analyte other than those to which the set of capture extender molecules bind;  
 (d) an optional preamplifier molecule comprising fifth and sixth hybridizing oligonucleotide segments, wherein the hybridizing oligonucleotide segments comprise a first nucleotidic unit which will not effectively base pair with A, T, C, G or U under conditions in which A-T and G-C base pairs are formed, and wherein the preamplifier molecule is capable of forming hybrid complexes with the label extender molecules and a plurality of amplification multimers;  
 (e) an amplification multimer comprising seventh and eighth hybridizing oligonucleotide segments, wherein the hybridizing oligonucleotide segments comprise a first nucleotidic unit which will not effectively base pair with A, T, C, G or U under conditions in which A-T and G-C base pairs are formed, and wherein the amplification multimer is capable of forming hybrid complexes with the label extender molecules or to the preamplifier molecules, and a plurality of identical oligonucleotide subunits; and  
 (f) label probes comprising a label, which are designed to form hybrid complexes with the identical oligonucleotide subunits and which provide, directly or indirectly, a detectable signal.  
 
     
     
         22 . The kit of    claim 21   , wherein the hybrid complexes are selected from the group consisting of T m1  hybrid complexes and T m2  hybrid complexes such that assay stringency can be controlled to selectively destabilize the T m1  hybrid complexes with the proviso that the label comprises a member of a T m2  complex.  
     
     
         23 . The kit of    claim 22   , wherein the T m1  hybrid complexes comprise at least one of the hybrid complexes selected from the group consisting of a capture probe/capture extender complex, a label extender/amplifier complex and a label extender/preamplifier complex, and wherein the label comprises a member of a T m2  hybrid complex.  
     
     
         24 . The kit of    claim 23   , wherein the T m2  hybrid complexes comprise one or more of the complexes selected from the group consisting of a label extender/amplifier complex, a label extender/preamplifier complex and a label/amplifier complex, with the proviso that the label comprises a member of a T m2  complex.  
     
     
         25 . An oligonucleotide useful as an aptamer, comprising an intramolecular oligonucleotide hybrid complex containing a plurality of complementary base pairs at least one of which comprises complementary nonnatural nucleotidic units that will not effectively base pair with adenosine (A), thymidine (T), cytidine (C), guanosine (G) or uridine (U) under conditions in which A-T and G-C base pairs are normally formed, and wherein the nonnatural nucleotidic unit is contained within an oligonucleotide segment in which specificity of the base pairs is not required for maintaining secondary structure of the aptamer.  
     
     
         26 . A method for preparing an aptamer comprising: 
 (a) providing a target molecule;    (b) contacting the target molecule with a randomer pool of oligonucleotides under conditions which favor binding of the oligonucleotides to the target molecule;    (c) separating the oligonucleotides which bind to the target molecule and form an oligonucleotide-target complex from the oligonucleotides which do not bind to the target molecule;    (d) dissociating the oligonucleotide from the oligonucleotide-target complex;    (e) amplifying the oligonucleotide using a polymerase chain reaction;    (f) repeating steps (b) through (e) at least once to form a final aptamer construct; and    (g) replacing one or more nucleotidic units in the final aptamer construct with nonnatural nucleotidic units that will not effectively base pair with adenosine (A), thymidine (T), cytidine (C), guanosine (G) or uridine (U) under conditions in which A-T and G-C base pairs are normally formed.    
     
     
         27 . An antisense molecule comprising first and second hybridizing segments, wherein the first hybridizing segment is capable of forming a hybrid complex with a target oligonucleotide and the second segment comprises at least one nucleotidic unit which will not effectively base pair with adenosine (A), thymidine (T), cytidine (C), guanosine (G) or uridine (U) under conditions in which A-T and G-C base pairs are formed, and is capable of forming a hybrid complex with a second antisense molecule.