Multi-chromophoric quencher constructs for use in high sensitivity energy transfer probes
View Patent ↗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. This can be accomplished by linking together, into a single molecule, two or more identical quenchers, by reacting the quenchers with a multivalent linker. The structure of the multi-chromophoric quencher can also be varied to quench a broader range of reporter emission wavelengths than previously possible. This can be accomplished by linking together, into a single molecule, two or more different quenchers, by reacting the quenchers with a multivalent linker. The structure of the multi-chromophoric quencher can also be varied to simultaneously broaden the absorption range and increase the total absorption within the absorption range. This can be done by combining the two concepts described above. In other words, 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 the following structural formula:
wherein:
each Q is diazoaryl dark quenching moiety selected from the group consisting of:
wherein the portion of the molecule contained within the dashed line box is a multivalent linking moiety L;
R y is H, C 1-4 alkyl, aryl or arylalkyl;
R is an acyclic moiety;
each LK is an amide moiety; and
R x is a reactive functional group.
2. The dark quencher construct of claim 1 , wherein each multivalent linking moiety is independently a multivalent monomeric moiety.
3. The dark quencher construct of claim 1 , further comprising a reactive functional group to facilitate attachment to one or more insoluble supports, probes for biomolecules, fluorescent dyes, or combinations thereof.
4. The dark quencher construct of claim 1 , wherein the reactive functional group is carboxyl.
5. The dark quencher construct of claim 1 , wherein L has between 1 and 20 non-hydrogen atoms selected from the group consisting of C, N and O.
6. The dark quencher construct of claim 1 , wherein the construct is formed by reacting a substituted amino acid with two quenchers.
7. The dark quencher construct of claim 1 , comprising the structure:
8. The dark quencher construct of claim 1 , wherein all of the dark quenching moieties are the same.
9. A dark quencher construct comprising the following structural formula:
wherein:
each Q is diazoaryl dark quenching moiety selected from the group consisting of:
wherein the portion of the molecule contained within the dashed line box is a multivalent linking moiety L;
R y is H, C 1-4 alkyl, aryl or arylalkyl;
R is an acyclic moiety;
each LK is independently selected from the group consisting of an ester, urea, carbonate, a carboxamide, a phosphate, an ether, a thioether, carbazide, hydrazine, a silane and a siloxane linkage; and
R x is a reactive functional group.
10. The dark quencher construct of claim 8 ; wherein the reactive functional group is selected from the group consisting of an ester, hydroxyl, a haloalkyl, a dienophile, an aldehyde, a ketone, a sulfonyl halide, a thiol, an amine, a Michael donor, a Michael acceptor, an epoxide, a cyanuryl halide, phosphoramidite, a substituted hydrazine, and a substituted diazyl alkane.