Oligonucleotide N3'-P5' thiophosphoramidates: their synthesis and use

Abstract

Oligonucleotides with a novel sugar-phosphate backbone containing at least one internucleoside 3'-NHP(O)(S<SUP>-</SUP>)O-5' linkage, and methods of synthesizing and using the inventive oligonucleotides are provided. The inventive thiophosphoramidate oligonucleotides were found to retain the high RNA binding affinity of the parent oligonucleotide N3'->P5' phosphoramidates and to exhibit a much higher acid stability.

1 . A polynucleotide comprising a non-homopolymeric sequence of nucleoside subunits joined by at least one inter-subunit linkage that is a N3′→P5′ thiophosphoramidate.  
     
     
         2 . The polynucleotide of  claim 1 , wherein the N3′→P5′ thiophosphoramidate linkages is defined by the formula:  
         3′-[—NH—P(═O)(—SR)—O—]-5′:  wherein R is selected from the group consisting of hydrogen, alkyl, aryl and salts thereof.    
     
     
         3 . The polynucleotide of  claim 2 , wherein all intersubunit linkages are N3′→P5′ thiophosphoramidate linkages.  
     
     
         4 . The polynucleotide of  claim 3 , wherein the defined sequence of the nucleoside subunits has a length of from 3 to 50.  
     
     
         5 . The polynucleotide of  claim 1 , comprising a second linkage selected from the group consisting of phosphodiester, phosphotriester, methylphosphonate, P3′→N5′ phosphoramidate, N3′→P5′ phosphoramidate, and phosphorothioate.  
     
     
         6 . The polynucleotide of  claim 5 , which is 3 to 50 nucleotide subunits in length.  
     
     
         7 . A polynucleotide comprising a sequence of nucleoside subunits containing at least one subunit defined by the formula:  
       
         
           
           
               
               
           
         
         wherein B is a purine or pyrimidine or an analog thereof;  
         Z is OR, SR, or methyl, wherein R is selected from the group consisting of hydrogen, alkyl, aryl and salts thereof; and  
         R 1  is selected from the group consisting of hydrogen, O—R 2 , S—R 2 , and halogen, wherein R 2  is H, alkyl, or (CH 2 ) n W(CH 2 ) m H, where n is between 1-10, m is between 0-10 and W is O, S, or NH, with the proviso that when Z is methy or OMe, R 1  is not H.  
       
     
     
         8 . The polynucleotide of  claim 7 , wherein all subunits are defined by the formula:  
       
         
           
           
               
               
           
         
       
     
     
         9 . The polynucleotide of  claim 7 , wherein the polynucleotide further comprises at least one subunit selected from the group consisting of phosphodiester, phosphotriester, methylphosphonate, P3′→N5′ phosphoramidate, N3′→P5′ phosphoramidate, and phosphorothioate subunits.  
     
     
         10 . The polynucleotide of  claim 7 , wherein each B moiety in the polynucleotide is independently selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 5-methylcytosine, 5-bromouracil, and inosine.  
     
     
         11 . The polynucleotide of  claim 7 , wherein the polynucleotide hybridizes with a target nucleic acid sequence.  
     
     
         12 . The polynucleotide of  claim 11 , wherein the target nucleic acid sequence is a sequence of telomerase RNA component.  
     
     
         13 . The polynucleotide of  claim 10 , wherein the polynucleotide can hybridize with an RNA target.  
     
     
         14 . The polynucleotide of  claim 7 , wherein the polynucleotide comprises a reporter moiety.  
     
     
         15 . The polynucleotide of  claim 14 , wherein the reporter moiety is selected from the group consisting of radioactive labels, biotin labels, and fluorescent labels.  
     
     
         16 . A method of synthesizing an oligonucleotide N3′→P5′ thiophosphoramidate, the method comprising: 
 (a) providing a first 5′-succinyl-3′-aminotrityl-2′,3′-dideoxy nucleoside attached to a solid phase support, the first nucleoside having a protected 3′ amino group;    (b) deprotecting the protected 3′ amino group to form a free 3′ amino group;    (c) reacting the free 3′ amino group with a 3′-protected aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer to form an internucleoside N3′→P5′ phosphoramidite linkage; and    (d) sulfurizing the internucleaside phosphoramidite group to form a N3′→P5′ thiophosphoramidate.    
     
     
         17 . A method according to  claim 16 , wherein reacting and sulferizing are repeated.  
     
     
         18 . A method of hybridizing polynucleotide to a DNA or RNA target comprising contacting a polynucleotide according to  claim 1  with the target under conditions that allow formation of a hybridization complex between the polynucleotide and the target.  
     
     
         19 . A method according to  claim 18 , wherein the polynucleotide carries a reporter moiety.  
     
     
         20 . A method according to  claim 19 , wherein the reporter moiety is selected from the group consisting of radioactive labels, biotin labels, and fluorescent labels.  
     
     
         21 . A method for determining a nucleic acid containing a specific sequence in a sample, comprising: 
 a) preparing a reaction mixture comprising the sample and a polynucleotide according to  claim 1  capable of hybridizing specifically with the sequence;    b) determining hybrids formed in the reaction mixture; and    c) correlating any hybrids formed with nucleic acid containing the specific sequence in the sample.    
     
     
         22 . A method for isolating a nucleic acid containing a specific sequence from a sample, comprising: 
 a) combining the sample and a polynucleotide according to  claim 1  capable of hybridizing specifically with the sequence; and    b) recovering the nucleic acid from hybrids formed with the polynucleotide.    
     
     
         23 . A method for inhibiting function of an RNA in a cell, comprising contacting the cell with a polynucleotide according to  claim 1  that can specifically hybridize with the RNA.  
     
     
         24 . A method according to  claim 23 , which is a method for inhibiting translation of an mRNA, wherein the polynucleotide comprises a sequence containing at least 10 bases complementary to a sequence contained in the mRNA.  
     
     
         25 . A method according to  claim 23 , which is a method for inhibiting telomerase enzyme in a cell, wherein the polynucleotide comprises a sequence complementary to telomerase RNA component.  
     
     
         26 . A method for inhibiting activity of a telomerase enzyme in a cell comprising contacting the cell with an effective amount of a polynucleotide according to  claim 1 .  
     
     
         27 . A kit for determining or isolating a nucleic acid containing a specific sequence in a sample, comprising a polynucleotide according to  claim 1  that can hybridize to the specific sequence, and written indications for using the polynucleotide for determining or isolating the nucleic acid.