Nucleotide analogs with six-membered rings

Abstract

The present invention is related to compounds comprising six-membered rings capable of being incorporated into nucleic acids. Particularly, the six-membered ring is a derivative of cyclohexane, cyclohexene, tetrahydropyran, tetrahydrothiopyran or piperidine. These compounds may be used to build up oligomeric compounds. The invention is further related to uses of these oligomeric compounds for hybridization and as probes. In addition, methods for the detection of nucleic acids are provided wherein the oligomeric compounds are used.

1 . An oligomeric compound comprising a monomeric unit having a structure of the formula  
       S-B-L-R 7    
     wherein S is a moiety comprising a six-membered ring, B is a heterocyclic base, L is a linking moiety, and R 7  is selected from the group consisting of a label, a protecting group or a solid phase.  
   
   
       2 . The compound according to  claim 1  wherein the six-membered ring is a derivative selected from the group consisting of cyclohexane, cyclohexene, tetrahydropyran, tetrahydrothiopyran or piperidine.  
   
   
       3 . An oligomeric compound comprising a monomer having a structure of the formula  
     
       
         
         
             
             
         
       
     
     wherein L is a linking moiety covalently and B is a heterocyclic base, 
 R 7  is selected from the group consisting of protecting group, a label, and a solid phase;  
 R 8  is selected from the group consisting of —H and —OR 11 ;  
 R 9  and R 11  selected from the group consisting of —H, a linking moiety covalently coupled to a solid phase, a phosphate, a phosphodiester with a nucleotide, a modified nucleotide, an oligonucleotide, and a modified oligonucleotide;  
 R 10  is selected from the group consisting of —H, —OH, alkyl, halogen, —O—R 5 , —S—R 5 , NR 5 R 5a , a label, and a linking moiety covalently coupled to a solid phase.  
 Y is selected from the group consisting of O, S, NR 5 , CR 5a R 5b , and CR 5a , and the bond between the C-1 atom of the six-membered ring and Y is a single bond when Y is O, S, NR 5 , or CR 5a R 5b  and a double bond when Y is CR 5a ; and  
 R 5  is selected from the group consisting of alkyl, alkenyl, alkinyl, aryl, acyl, a protecting group, and —H and R 5a  and R 5b  are selected from the group consisting of alkyl, alkenyl, alkinyl, aryl, acyl, and —H.  
 
   
   
       4 . The compound according to  claim 3  wherein the heterocyclic base is selected from the group consisting of adenine, guanine, cytosine, thymine, uracil, and methyl-cytosine.  
   
   
       5 . The compound according to  claim 3  wherein the linking moiety L comprises carbon, oxygen or nitrogen atoms and a reactive group.  
   
   
       6 . The compound according to  claim 3  wherein R 7  is a label selected from the group consisting of a dye and a hapten.  
   
   
       7 . The compound according to  claim 6  wherein the dye is selected from the group consisting of a fluorescein dye, a rhodamine dye, a cyanine dye, a coumarin dye, and an azo dye.  
   
   
       8 . The compound according to  claim 6  wherein the hapten is biotin.  
   
   
       9 . The compound according to  claim 3  wherein R 8  is —OR 11 , R 9  and R 11  are an oligonucleotide or a modified oligonucleotide, and R 10  is —H or —OH.  
   
   
       10 . The compound according to  claim 3  wherein Y is selected from the group consisting of O, S, and CR 52 R 5b .  
   
   
       11 . The compound according to  claim 3  wherein Y is CR 5a  and R 5b  is selected from the group consisting of alkyl, alkenyl, alkinyl, aryl, acyl, and —H.  
   
   
       12 . A composition for analyzing interactions between target nucleic acids and oligomeric compounds comprising an array of a plurality of oligomeric compounds having different sequences wherein the oligomeric compound is an oligomeric compound according to  claim 3  wherein a solid phase is covalently coupled to a member selected from the group consisting of R 7 , R 9 , R 10 , and R 11 .  
   
   
       13 . A method for determining the presence or amount of a target nucleic acid in a sample comprising the steps of: 
 providing a sample suspected of containing the target nucleic acid,    providing a compound according to  claim 1  or  3 , wherein said compound is essentially complementary to a part or all of the target nucleic acid,    optionally amplifying the target nucleic acid with a template-dependent DNA polymerase and primers,    contacting the sample with the compound under conditions for binding the compound to the target nucleic acid,    determining the binding product or the degree of hybridization between the target nucleic acid and the compound as a measure of the presence, absence or amount of the target nucleic acid.    
   
   
       14 . The method according to  claim 13  whereby the degree of hybridization is determined by the quantity of the first or second fluorescent label that is released from the oligomeric compound hybridized to the target nucleic acid by exnuclease hydrolysis by the template-dependent DNA polymerase.  
   
   
       15 . The method according to  claim 14  further comprising the steps of: 
 performing a cycling step comprising an amplifying step and a hybridizing step, wherein the amplifying step comprises contacting the sample with primers to produce a an amplification product if target nucleic acid is present in said sample, wherein the hybridizing step comprises contacting the sample with a pair of probes, wherein at least one of the probes is an oligomeric compound comprising a monomeric unit comprising a six-membered ring, wherein the members of the pair of probes hybridize to the amplification product within no more than five nucleotides of each other, wherein the first probe of the pair of probes is labeled with a donor fluorescent label and wherein a second probe of the pair of probes is labeled with a corresponding acceptor fluorescent label, and    detecting the presence or absence of fluorescence resonance energy transfer between the donor fluorescent label of the first probe and the acceptor fluorescent label of the second probe, wherein the presence of fluorescence resonance energy transfer is indicative of the presence of the target nucleic acid in the sample, and wherein the absence of fluorescence resonance energy transfer is indicative of the absence of the target nucleic acid in the sample.    
   
   
       16 . A method for determining the presence or amount of a target nucleic acid in a sample comprising the steps of: 
 providing a target nucleic acid from a sample to be analyzed;    synthesizing double stranded complementary DNA from the target nucleic acid,    amplifying the target nucleic acid in the presence of a compound according to  claim 1  and nucleoside triphosphates whereby a labelled nucleic acid is obtained,    hybridizing the labelled target nucleic acid to an array of oligomeric compounds at a defined location, and    measuring the fluorescence intensities at each defined location whereby the presence or the amount of the target nucleic acid is determined.    
   
   
       17 . A kit for determining a target nucleic acid in a sample comprising a template-dependent polymerase, a set of primers, nucleotides, and an oligomeric compound according to  claim 1 , wherein R 7  is a label.  
   
   
       18 . A kit for determining a target nucleic acid in a sample comprising a template-dependent polymerase, a set of primers, nucleotides, and an oligomeric compound according to  claim 3 , wherein R 7  is a label.