Fluorescent quenching detection reagents and methods

Abstract

Oligonucleotide probes containing two labels are provided and are useful in hybridization assays. The probes can also contain a minor groove binding group.

1 . An oligonucleotide probe compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 Ar 1  and Ar 2  are each independently a substituted or unsubstituted aryl group, and one of Ar 1  and Ar 2  is directly or indirectly substituted with a substituted aryl group (Ar 3 ), where Ar 3  extends the resonance ability of the Ar 1 —N═N—Ar 2  aromatic system and thereby increases the wavelength absorbance maximum of the compound;  
 MGB is a minor groove binding group;  
 FL is a fluorescent group having an emission maxima in the region from about 400 to about 900 nm;  
 K is a cyclic or acyclic linking group having from 1 to 30 backbone atoms selected from C, N, O, S and P;  
 W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof;  
 [A—B] n  is a natural or modified oligonucleotide where the subscript n is an integer of from 4 to 100; and  
 the subscript zz is 0 or 1.  
 
     
     
         2 . A quencher-phosphoramidite reagent compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 Ar 1  and Ar 2  are each independently a substituted or unsubstituted aryl group, and one of Ar 1  and Ar 2  is directly or indirectly substituted with a substituted aryl group (Ar 3 ), where Ar 3  extends the resonance ability of the Ar 1 —N═N—Ar 2  aromatic system and thereby increases the wavelength absorbance maximum of the compound;  
 X 1  is selected from the group consisting of H, (C 1 -C 12 )alkyl, aryl, heteroaryl, and protected or unprotected functional group;  
 X 2  is a moiety reactive towards nucleophiles; and  
 W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof.  
 
     
     
         3 . A compound of  claim 2  wherein X 2  is selected from the group consisting of a phosphorous coupling moiety, a pentafluorophenoxy moiety and a succinimidyl moiety.  
     
     
         4 . A compound of  claim 2  wherein X 1  is selected from the group consisting of OH, O-dimethoxytrityl, O-methoxytrityl, O-trityl or an oxygen atom having an acid labile blocking group.  
     
     
         5 . A compound of  claim 2  wherein X 2  is a phosphoramidite of the formula —O—P(N(iPr) 2 )(OCH 2 CH 2 CN).  
     
     
         6  A compound of claims  1  or  2  wherein Ar 2  is substituted with Ar 3 .  
     
     
         7  A compound of  claim 6  wherein Ar 2  is indirectly substituted with Ar 3 .  
     
     
         8 . A compound of  claim 7  wherein Ar 2  is indirectly substituted with Ar 3  through aromatic ring(s) and/or double bond(s).  
     
     
         9 . A compound of  claim 8  wherein Ar 2  is indirectly substituted with Ar 3  through a double bond selected from carbon-carbon and nitrogen-nitrogen double bonds.  
     
     
         10 . A compound of  claim 9  wherein Ar 2  is indirectly substituted with Ar 3  through a group selected from —(C≡C) t — and —(CR′═CR′) t — where t is 0 to 5 and R′ is independently selected from hydrogen, (C 1 -C 8 )alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C 1 -C 4 )alkyl, and (unsubstituted aryl)oxy-(C 1 -C 4 )alkyl.  
     
     
         11 . A compound of claims  1  or  2  wherein at least one of Ar 1 , Ar 2  and Ar 3  is substituted with -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO 2 , —CO 2 R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O) 2 R′, —NR′—C(O)NR″R′″, —NH—C(NH 2 )=NH, —NR′C(NH 2 )=NH, —NH—C(NH 2 )=NR′, —S(O)R′, —S(O) 2 R′, —S(O) 2 NR′R″, —N 3 , —CH(Ph) 2 , perfluoro(C 1 -C 4 )alkoxy, and perfluoro(C 1 -C 4 )alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R″ and R′″ are independently selected from hydrogen, (C 1 -C 8 )alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C 1 -C 4 )alkyl, and (unsubstituted aryl)oxy-(C 1 -C 4 )alkyl.  
     
     
         12 . A compound of  claim 11  wherein Ar 2  is indirectly substituted with Ar 3  through a double bond.  
     
     
         13  A compound of  claim 12 , wherein Ar 2 —N═N—Ar 1 — has the structure:  
       
         
           
           
               
               
           
         
       
     
     
         14 . A compound of  claim 11  wherein Ar 2  is directly substituted with Ar 3 .  
     
     
         15 . A compound of  claim 14  wherein Ar 2 —N═N—Ar 1 — has the structure:  
       
         
           
           
               
               
           
         
       
     
     
         16 . An oligonucleotide probe compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 Ar 1  and Ar 2  are each independently a substituted or unsubstituted aryl group;  
 MGB is a minor groove binding group;  
 FL is a fluorescent group having an emission maxima in the region from about 400 to about 900 nm;  
 K is a cyclic or acyclic linking group having from 1 to 30 backbone atoms selected from C, N, O, S and P;  
 W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof, where W modulates the absorption wavelength of the Ar 1 —N═N—Ar 2  moiety to increase the wavelength absorbance of the compound;  
 [A—B] n  is a natural or modified oligonucleotide where the subscript n is an integer of from 4 to 100; and  
 the subscript zz is 0 or 1.  
 
     
     
         17 . A quencher-phosphoramidite reagent compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 Ar 1  and Ar 2  are each independently a substituted or unsubstituted aryl group;  
 X 1  is selected from the group consisting of H, (C 1 -C 12 )alkyl, aryl, heteroaryl, and protected or unprotected functional group;  
 X 2  is a moiety reactive towards nucleophiles; and  
 W is a linking group having from 3 to 100 backbone atoms selected from C, N, O, S, Si and P, said linking group being cyclic, acyclic, aromatic or a combination thereof, where W modulates the absorption wavelength of the Ar 1 —N═N—Ar 2  moiety to increase the wavelength absorbance of the compound.  
 
     
     
         18 . A compound of  claim 17  wherein X 2  is selected from the group consisting of a phosphorous coupling moiety, a pentafluorophenoxy moiety and a succinimidyl moiety.  
     
     
         19 . A compound of claims  16  or  17  wherein W comprises a substituted aryl group (Ar 3 ) which is directly or indirectly attached to Ar 1  and extends the resonance ability of the Ar 1 —N═N—Ar 2  aromatic system and thereby increases the wavelength absorbance maximum of the compound.  
     
     
         20 . A compound of  claim 19  wherein Ar 1  is indirectly substituted with Ar 3  through aromatic ring(s) and/or double bond(s).  
     
     
         21 . A compound of  claim 20  wherein Ar 1  is indirectly substituted with Ar 3  through a double bond selected from carbon-carbon and nitrogen-nitrogen double bonds.  
     
     
         22 . A compound of  claim 21  wherein Ar 1  is indirectly substituted with Ar 3  through a group selected from —(C≡C) t — and —(CR′═CR′) t — where t is 0 to 5 and R′ is independently selected from hydrogen, (C 1 -C 8 )alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C 1 -C 4 )alkyl, and (unsubstituted aryl)oxy-(C 1 -C 4 )alkyl.  
     
     
         23 . A probe compound-for a hybridization assay comprising the formula FL-ODN-(W) d -Q wherein 
 FL is a fluorophore with an emission wavelength in the range of about 300 to about 800 nm;    ODN is an oligonucleotide;    W is a linker group that has from 3 to 100 atoms other than hydrogen atoms, selected from C, N, O, S, P and Si, and is cyclic, acyclic, aromatic or a combination thereof, and d is 0 or 1; and    Q is a quencher moiety comprising the structure —Ar 1 —N═N—Ar 2  wherein Ar 1  is joined to either ODN or W, and Q has a broader absorbance range than dabcyl (4-{[4-(dimethylamino)phenyl]diazenyl}benzoyl, absorbance max=453 nm), wherein Ar 1  and Ar 2  are each independently a substituted or unsubstituted aryl group.    
     
     
         24 . A compound of  claim 23  wherein one of Ar 1  and Ar 2  is directly or indirectly substituted with a substituted aryl group (Ar 3 ), where Ar 3  extends the resonance ability of the Ar 1 —N═N—Ar 2  aromatic system and thereby increases the wavelength absorbance maximum of the compound.  
     
     
         25 . A compound of the formula  
       
         
           
           
               
               
           
         
       
       wherein  
       
         
           
           
               
               
           
         
         L is a cleavable linker;  
         q is 0 or 1;  
         MGB is a minor groove binder;  
         r is 0 or 1;  
         W is a linker group that has from 3 to 100 atoms other than hydrogen atoms, selected from C, N, O, S, P and Si, and is cyclic, acyclic, aromatic or a combination thereof;  
         Q is a quencher moiety comprising the formula —Ar 1 —N═N—Ar 2  where Ar 1  is joined to W and wherein Ar 1  and Ar 2  are each independently a substituted or unsubstituted aryl group, and one of Ar 1  and Ar 2  is directly or indirectly substituted with a substituted aryl group (Ar 3 ), where Ar 3  extends the resonance ability of the Ar 1 —N═N—Ar 2  aromatic system and thereby increases the wavelength absorbance maximum of the compound; and  
         Y is selected from 
 —O—J 1  where J 1  is a hydroxyl protecting group;  
 an oligonucleotide (-ODN); and  
 an oligonucleotide-fluorophore conjugate (-ODN-FL).  
 
       
     
     
         26 . A compound of claims  24  or  25  wherein Ar 2  is indirectly substituted with Ar 3  through aromatic ring(s) and/or double bond(s).  
     
     
         27 . A compound of  claim 26  wherein Ar 2  is indirectly substituted with Ar 3  through a double bond selected from carbon-carbon and nitrogen-nitrogen double bonds.  
     
     
         28 . A compound of claims  24  or  25  wherein W comprises Ar 3 , and Ar 1  is indirectly substituted with Ar 3  through aromatic ring(s) and/or double bond(s).  
     
     
         29 . A compound of  claim 28  wherein Ar 1  is indirectly substituted with Ar 3  through a double bond selected from carbon-carbon and nitrogen-nitrogen double bonds.  
     
     
         30 . A compound of  claim 25  wherein q is 0.  
     
     
         31 . A compound of  claim 25  wherein the solid support is selected from glass, controlled pore glass, polymeric materials, polystyrene, beads, coated glass or support that is compatible with oligonucleotide synthesis.