Nucleic acid probes and methods

Abstract

The present invention provides metal-containing purines, pyrimidines, nucleosides, nucleotides and oligonucleotides; including phosphoramidite and photolabile derivatives thereof, including methods of making and method of using same. The present invention provides a method for detection of nucleic acid sequences via electrochemical or photochemical means.

We claim:  
     
         1 . A nucleoside, nucleotide, purine or pyrimidine derivative comprising a detectable marker which is a metal complex containing at least one of a M(diimine) x   Y+  complex, M(terpyridine) x   Y+  complex or a metallocene wherein M is a transition metal, said diimine is selected from a bipyridine, phenanthroline or terpyridine derivative; x is 1, 2, or 3 and y is 0, 1, 2, 3, or 4, said bipyridine, phenanthroline, terpyridine and metallocene being optionally substituted by any one of alkyl, alkene, alkyne, aryl, alkylaryl, and carboxyalkyl, amide, ester or ether.  
     
     
         2 . A nucleoside, nucleotide, purine or pyrimidine derivative according to  claim 1  wherein said detectable marker contains at least one of a M(diimine) 2 (diimine) 2+  complex wherein M is a metal selected from Fe +2 , Ru +2 , Os +2 , Co +2 , Rh +2,  and Cr +2 , or a M(diimine) 2 (diimine) 3+  complex wherein M is a metal selected from Fe +3 , Ru +3 , Os +3 , Co +3 , Rh +3 , and Cr +3 , or a M(terpyridine) 2   3+  complex wherein M a metal selected from Fe +3 , Ru +3 , Os +3 , Co +3 , Rh +3 , and Cr +3 , or a M(diimine) 2   3+  complex wherein M is a metal selected from Fe +2 , Ru +2 , Os +2 , CO +2 , Rh −2 , and Cr +2 .  
     
     
         3 . An oligonucleotide comprising at least one nucleoside, nucleotide or nucleic acid derivative of  claim 1 .  
     
     
         4 . A nucleoside, nucleotide, purine or pynimidine derivative of  claim 1  which comprises a sugar selected from the group consisting of a ribose, a deoxyribose and a dideoxyribose.  
     
     
         5 . A nucleoside nucleotide, purine or pyrimidine derivative of  claim 1  which fuirther comprises a dimethoxytritylchloride, α-methyl-6-nitropipronyloxy carbonyl or 2-cyanoethyl N,N′-diisopropylcholoro phosphoramidite derivative.  
     
     
         6 . A nucleoside, nucleotide, purine or pyrimidine derivative of  claim 1  which further comprises a compound of the following structure:  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3 , and R 4  independently are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl. thio, thioether, amino, nitro, carboxyl, formate, foramino or phosphido group, or adjacent substituents R 1 -R 4  are substituted oxygen groups that together form a cyclic acetal or ketal; R 5  is hydrogen, alkoxyl, alkyl, halo, aryl or alkenyl group, n=0, 1, 2 or 3; and Y is a hydroxyl group of the nucleoside or nucleotide.  
     
     
         7 . A nucleotide, or nucleoside, purine or pyrimidine derivative of  claim 6 , wherein Y is a 5′-hydroxyl or a 3′-hvdroxyl group of said nucleoside or nucleotide.  
     
     
         8 . A double-stranded nucleic acid sequence comprising the oligonucleotide of  claim 3  and an electron acceptor moiety which contains a metal selected from the group consisting of Cr, Cu, Co, Fe, Ru and Os.  
     
     
         9 . A nucleoside, nucleotide, purine or pvrimidine derivative of  claim 1  which contains a uricil, adenine, thymine, cytosine, guanine or natural or synthetic analogs or derivatives thereof.  
     
     
         10 . A nucleoside, nucleotide, purine or pyrimidine derivative of  claim 1  which contains a di- or tri-phosphate optionally substituted by a sulfur, NH or BH group.  
     
     
         11 . A nucleoside, nucleotide, purine or pyrimidine according to  claim 1 , or an oligonucleotide of  claim 3  attached to a solid surface through one of a covalent bond, an electrostatic association or hydrogen bond.  
     
     
         12 . A nucleoside, nucleotide, purine or pyrimidine according to  claim 11  wherein said solid surface is one of a nanoparticle, glass, ITO, TiO 2 , SnO 2 , Au, Pt, silicon, porous silicon, plastic or graphite.  
     
     
         13 . A nucleoside, nucleotide, purine or pyrimidine derivative according to  claim 1  wherein said detectable marker is selected from a bis(2,2′-bipyridine)(4′-methyl-2,2′-bipyridine-4-carbonyl propargyl amine) ruthenium (II) substituent, a ferrocene substituent; and a bis(2,2′-bipyridine) (4′-methyl-2,2′-bipyridine-4-carbonyl propargyl amine) osmium (II) substituent.  
     
     
         14 . A nucleoside, purine or pyrimidine nucleotide of  claim 1  wherein said complex is chiral wherein said chirality is fac or ras, R or S, or D or L.  
     
     
         15 . A nucleoside, nucleotide, purine or pyrimidine derivative according to  claim 1  wherein said diimine ligands around said metal are all the same.  
     
     
         16 . A nucleoside, nucleotide, purine or pyrimidine derivative according to  claim 1  wherein at least two of said diimine ligands around said metal are not the same.  
     
     
         17 . A method of identifying a nucleic acid molecule comprising contacting said nucleic acid molecule with an oligonucleotide of  claim 3  under conditions where said oligonucleotide can specifically bind to complementary regions of said nucleic acid to form an identifiable complex, and detecting said binding.  
     
     
         18 . A method according to  claim 17 , wherein said method is an in vivo, ex vivo or in vitro method.  
     
     
         19 . A method according to  claim 17 , wherein said detecting comprises measuring a photophysical alteration or an electrochemical event of said complex.  
     
     
         20 . A method according to  claim 19  wherein said photophysical alteration is an energy or electron transfer event.  
     
     
         21 . A method according to  claim 17  wherein said identifiable complex is a double or triple helix.  
     
     
         22 . A method according to  claim 17  wherein said nucleic acid molecule is of human, animal, viral, bacteria, or plant origin.  
     
     
         23 . A method according to  claim 19  wherein said electron transfer event occurs due to interaction between two metal complexes on a single nucleic acid strand.  
     
     
         24 . A method according to  claim 19  wherein said electron transfer event occurs due to interaction between two metal complexes on a separate nucleic acid strand.  
     
     
         25 . A method according to  claim 19  wherein said electron transfer event occurs due to interaction between a metal complex on a single nucleic acid strand and said nucleoside or nucleotide.  
     
     
         26 . A method according to  claim 19  wherein said electron transfer event occurs due to an interaction between a metal complex and an organic donor/acceptor on a single nucleic acid strand 
 wherein said organic donor/acceptor is selected from the group comprising acridine, fluorescein, anthracene, metylphenothiazine, methylviolgen, phenothiazine and quione.  
 
     
     
         27 . A method according to  claim 19  wherein said detection is between a metal complex on a single strand and an organic donor/acceptor on a second single nucleic acid strand wherein said organic donor/acceptor is selected from the group comprising acridine, fluorescein, anthracene, metylphenothiazine, methylviolgen, and quione.  
     
     
         28 . A method according to  claim 19  wherein the electron transfer event is a photoinduced electron transfer event, electrochemical event, or an electroluminescence event.  
     
     
         29 . A method according to  claim 28  wherein the electron-transfer event is performed electrochemically.  
     
     
         30 . A method according to  claim 17  wherein said oligonucleotide is attached to a surface and said metal complex donates an electron or energy to the surface or said metal complex accepts and electron energy from the surface, and said detecting comprising measuring a transfer of said electron or energy.  
     
     
         31 . A method according to  claim 17  wherein said oligonucleotide binds directly or indirectly to a nucleic acid molecule attached to a surface wherein said metal complex donates an electron or energy to the surface or said metal complex accepts and electron or energy from the surface and said detecting comprising measuring a transfer of said electron or energy.  
     
     
         32 . An improved method of synthesizing a labeled oligonucleotide comprising: 
 attaching a nucleoside of the oligonucleotide to be synthesized to a solid support through a labile linker arm;    said nucleoside containing a removable protecting group attached to a 5′hyroxyl of a sugar group of said nucleoside;    removing said protecting group;    adding a further nucleoside containing a labile linker arm and a protecting group to said solid support under conditions where said further nucleoside is coupled to said 5′hydroxyl of said sugar group through said linker arm;    optionally capping any uncoupled nucleoside;    removing the protecting group of said further nucleoside;    repeating the above with further nucleoside derivatives wherein at least one of said further nucleoside contains a detectable label, until a desired oligonucleotide is formed; and    cleaving said oligonucleotide from said solid support;    wherein the improvement comprises adding at least one dimethoxytrityl, phosphoramidite halodonucleoside derivative in place of one of said at least one nucleoside containing a detectable label; and    adding a detectable label to said halodonucleoside derivative subsequent to formation of said oligonucleotide.    
     
     
         33 . The method of  claim 32  wherein said cleaving comprising adding concentrated ammonium hydroxide.  
     
     
         34 . The method of  claim 32 , further comprising heating said cleaved oligonucleotide to remove any protecting groups.  
     
     
         35 . An oligonucleotide comprising at least one nucleoside, nucleotide or nucleic acid of  claim 2 .  
     
     
         36 . An improved method of oligonucleotide synthesis comprising combining phosphoramidite nucleoside derivatives in combination with a solid support wherein the improvement comprises adding at least one phosphoramidite halonucleoside during the synthesis and subsequently labeling said halonucleoside with a marker in a metal catalyzed reaction.  
     
     
         37 . The improved method of  claim 36  wherein said metal is paladium.  
     
     
         38 . The improved method of  claim 36  wherein said halonucleoside is selected from the group consisting of fluorouridine, fluoroadenosine, fluoroguanosine, fluorocytidine, fluorothymidine, chlorouridine, chloroadenosine, chloroguanosine, chlorocytidine, chlorothymidine, iodouridine, iodoadenosine, jodoguanosine, iodocytidine, iodothymidine, bromouridine, bromoadenosine, bromoguanosine, bromocytidine and bromothymidine.  
     
     
         39 . The improved method of  claim 36  wherein said oligonucleotide synthesis progresses in the 3′ to 5′ or the 5′ to 3′ direction.  
     
     
         40 . The improved method of  claim 36  wherein the marker is a metal-containing alkylene derivative.