Multi-Chromophoric Quencher Constructs for Use in High Sensitivity Energy Transfer Probes

Abstract

Dark quencher constructs, termed “multi-chromophoric quenchers” are described herein that comprise at least two dark quenching moieties, which can be the same or different, linked together by at least one multivalent linking moiety. The structure of the multi-chromophoric quenchers can be varied to selectively enhance quenching within a specific range of reporter emission wavelengths, to quench a broader range of reporter emission wavelengths than previously possible, or can combine both concepts. Multiple types of quenching moieties can be employed to increase the absorption range and a multiple number of each type of quenching moiety can be used to increase the total absorptivity within the absorption range. The multi-chromophoric quenchers can be tethered to probes for biomolecules, insoluble supports and/or fluorescent dyes for use in a wide variety of biomolecular assays.

1 . A dark quencher construct comprising at least two dark quenching moieties, wherein a first dark quenching moiety and a second dark quenching moiety of the at least two dark quenching moieties have different structures, have different absorption ranges, and are linked together by at least one multivalent linking moiety. 
     
     
         2 - 3 . (canceled) 
     
     
         4 . The dark quencher construct of  claim 1  in which at least one of the at least two dark quenching moieties comprises a group selected from diazoaryl, bisdiazoaryl, aminanthraquinone, bis-n-phenyl-rhodamine, 4,5 dihydroxyfluorescein, and 4,5-dialkoxyfluorescein. 
     
     
         5 . The dark quencher construct of  claim 1  in which at least one of the at least two dark quenching moieties comprises a group selected from the following: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         where L represents a multivalent linkage moiety, R y  is H, C1-4 alkyl, aryl, or arylalkyl and each dashed line indicates point of attachment for the remainder of the dark quencher construct. 
       
     
     
         6 . The dark quencher construct of  claim 1  where the multivalent linking moiety is selected from multivalent monomelic, linear polymeric, branched polymeric and dendritic backbones. 
     
     
         7 . The dark quencher construct of  claim 1  where the said quenching moieties are pendant from a single multivalent linking moiety. 
     
     
         8 . The dark quencher construct of  claim 1  where each quenching moiety is separated by a multivalent linking moiety in linear fashion within a linear or branched oligomeric or polymeric backbone. 
     
     
         9 . The dark quencher construct of  claim 1  further comprising one or more reactive functional groups to facilitate attachment to one or more insoluble supports, probes for biomolecules, fluorescent dyes, or combinations thereof. 
     
     
         10 . The dark quencher construct of  claim 9  where a reactive functional group is pendant from the multivalent linking moiety. 
     
     
         11 . The dark quencher construct of  claim 9  where a reactive functional group is pendant from a quencher moiety. 
     
     
         12 . The dark quencher construct of  claim 9  where each reactive functional group is selected from carboxyl groups, esters, hydroxyl groups, haloalkyl groups, dienophile groups, aldehyde groups, ketone groups, sulfonyl halide groups, thiol groups, amine groups, Michael donors, Michael acceptors, epoxides, cyanuryl halides, phosphoramidites, substituted hydrazines, and substituted diazyl alkanes. 
     
     
         13 - 14 . (canceled) 
     
     
         15 . The dark quencher construct of  claim 16  comprising the following structure: 
       
         
           
           
               
               
           
         
         where each Q is selected from the first dark quenching moiety and the second dark quenching moiety. 
       
     
     
         16 . The dark quencher construct of  claim 1  comprising the following structure: 
       
         
           
           
               
               
           
         
         where each Q is selected from the first dark quencher moiety and the second dark quencher moiety; where R x  is. a reactive functional group; where the remainder of the molecule (contained within the dashed line box) is a multivalent linking moiety where R is an acyclic, aryl or alkylaryl moiety and each LK, independently, is a moiety that comprises one or more ester, urea, urethane, carbamate, amide, amine, phosphate, sulfonamide, ether, thioether, carbazide, hydrazone, silane, and siloxane linkages. 
       
     
     
         17 . The dark quencher construct of  claim 16  formed by reacting a substituted amino acid with two quenchers. 
     
     
         18 - 33 . (canceled) 
     
     
         34 . A conjugate comprising a carrier portion comprising a probe for a biomolecule and at least one dark quenching portion, wherein each dark quenching portion is a construct of  claim 1 . 
     
     
         35 - 40 . (canceled) 
     
     
         41 . The conjugate of  claim 34  wherein the probe for a biomolecule is selected from an amino acid, a peptide, a peptide analog, a protein, a carbohydrate, a polynucleotide, a polynucleotide analog, hormones, an antigen, an antibody and combinations thereof. 
     
     
         42 . The conjugate of  claim 34  wherein the carrier portion comprises a fluorescent dye that forms a donor-acceptor energy transfer pair with at least one dark quenching moiety on the dark quenching portion. 
     
     
         43 . The conjugate of  claim 42  wherein the fluorescent dye is selected from fluoresceins, rhodamines, rhodols, cyanines, napthylamines, acridines, benzoxadiazoles, stilbenes, pyrenes, pyronines, coumarins, and porphyrins. 
     
     
         44 - 46 . (canceled) 
     
     
         47 . The conjugate of  claim 42  wherein the fluorescent dye and/or the quenching portion are attached to the probe by a linking group that is cleaved upon interaction with an analyte. 
     
     
         48 . The conjugate of  claim 34  wherein the carrier portion comprises a probe for a biomolecule, where said probe is a nucleic acid which can, optionally, be modified at any base moiety, sugar moiety or phosphate backbone to create nucleic acid analogs or contain additional groups. 
     
     
         49 . The conjugate of  claim 48  wherein the carrier portion additionally comprises a fluorescent dye. 
     
     
         50 . The conjugate of  claim 49  wherein said probe is a nucleic acid with a stem loop architecture that straightens upon hybridization with a target, thereby separating the quencher moieties and fluorescent dye. 
     
     
         51 . The conjugate of  claim 49  wherein the probe is a nucleic acid that is cleaved upon interaction with a target. 
     
     
         52 - 58 . (canceled) 
     
     
         59 . The conjugate of  claim 34  wherein the carrier portion comprises a probe for a biomolecule and a fluorescent dye, where the probe is a peptide that contains a cleavage recognition site for an enzyme. 
     
     
         60 . The conjugate of  claim 59  wherein the peptide contains a cleavage recognition site for a protease. 
     
     
         61 . The conjugate of  claim 59  wherein the cleavage recognition site is for an enzyme selected from trypsin, enterokinase, HIV-I protease, prohormone convertase, interleukin-1b-converting enzyme, adenovirus endopeptidase, cytomegalovirus assemblim, leishmanolysin, β-secretase for amyloid precursor protein, thrombin, renin, angiotensin-converting enzyme, cathepsin-D and a kininogenase. 
     
     
         62 - 63 . (canceled) 
     
     
         64 . A method of synthesizing a multi-chromophoric dark quencher molecule of  claim 1  having a broadened energy absorption profile comprising the following steps:
 (i) selecting multiple dark quencher molecules that have different absorption spectrums, where said dark quencher molecules have at least one reactive functional group; and 
 (ii) linking said dark quencher molecules to a linking molecule that comprises reactive functional groups capable of reacting with the reactive functional groups on the dark quencher molecules to form linkages, 
 where the absorption spectrum of the multi-chromophoric dark quencher molecule is broader than the absorption spectrum of its individual dark quencher molecule components. 
 
     
     
         65 . (canceled)