Non-fluorescent quencher compounds and biomolecular assays
AssigneeApplied Biosystems, LLC
InventorsGregory J. Ewing, Khairuzzaman Bashar Mullah, Ronald Graham
Life of this patent
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Abstract
Bis-diazo, triaryl and aryldiazo-N-arylphenazonium quencher moieties, substituted with electron-withdrawing and electron-donating substituents which induce polarity in the delocalized aryl/diazo ring systems, are useful as labels when attached to biomolecules such as polynucleotides, nucleosides, nucleotides and polypeptides. The quencher moieties are non-fluorescent and accept energy transfer from fluorescent reporter labels by any energy-transfer mechanism, such as FRET. Fluorescence quencher compositions are useful in preparing quencher labeled biomolecules for various molecular biology assays based on fluorescence detection.
Claims29
A composition useful for synthesizing a quencher-labeled polynucleotide which has the structure (II):
- wherein:
- Q is a quencher moiety Q1 having the following structure:
- wherein:
- R1, R2, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 and R14 are each, independently of one another, selected from hydrogen, an electron-withdrawing group and an electron-donating group, or, alternatively R4 may be taken together with R5 or R6 may be taken together with R7 to form a benzo group,
- further wherein:
- at least one of R1, R2, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 or R14 is an electron-withdrawing group;
- and
- at least one of R1, R2, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 or R14 is an electron-donating group;
- and
- a reactive linking group linking the aryl carbon of the quencher moiety to L1, either directly or by way of a linker NR″R″ moiety, where each R″ is independently C1-C12 alkyl;
- L1, L3 and L4 are each, independently of one another, selected from a bond, C1-C12 alkyldiyl, C1-C12 alkoxyldiyl, C1-C12 alkylaminodiyl, C1-C12 alkylamidediyl, C5-C14 aryldiyl, and 1-20 ethyleneoxy units;
- L2 is a C1-C12 alkoxyldiyl, wherein the hydroxyl group is attached to X;
- A is a cleavable linker;
- X is an acid-labile protecting group;
- Y is selected from N and CR, where R is selected from H, C1-C6 alkyl, and C5-C14 aryl;
- and
- represents the solid support.
A compound having a structure of one of the following formulae:
A compound having a structure of one of the following formulae:
A 3′ quencher polynucleotide bound to a solid support, having the structure:
- wherein the polynucleotide comprises 2 to 100 nucleotides;
- Q is a quencher moiety Q1 having the following structure:
- wherein:
- R1, R2, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 and R14 are each, independently of one another, selected from hydrogen, an electron-withdrawing group and an electron-donating group, or, alternatively R4 may be taken together with R5 or R6 may be taken together with R7 to form a benzo group, further wherein:
- at least one of R1, R2, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 or R14 is an electron withdrawing group;
- and
- at least one of R1, R2, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 or R14 is an electron donating group;
- and
- a reactive linking group linking the aryl carbon of the quencher moiety to L1, either directly or by way of a linker NR″R″ moiety, where each R″ is independently C1-C12 alkyl;
- L1, L3 and L4 are each, independently of one another, selected from a bond, C1-C12 alkyldiyl, C1-C12 alkoxyldiyl, C1-C12 alkylaminodiyl, C1-C12 alkylamidediyl, C5-C14 aryldiyl, and 1-20 ethyleneoxy units;
- L2 is a C1-C12 alkoxyldiyl, wherein the hydroxyl group is attached to a phosphodiester moiety of a first nucleotide;
- A is a cleavable linker;
- Y is selected from N and CR, where R is selected from H, C1-C6 alkyl, and C5-C14 aryl;
- and
- represents the solid support.
A 5′ quencher polynucleotide bound to a solid support, having the structure:
- wherein the polynucleotide comprises 2 to 100 nucleotides;
- Q is a quencher moiety Q1 having the following structure:
- wherein:
- R1, R2, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 and R14 are each, independently of one another selected from hydrogen, an electron-withdrawing group and an electron-donating group, or, alternatively R4 may be taken together with R5 or R6 may be taken together with R7 to form a benzo group, further wherein:
- at least one of R1, R2, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 or R14 is an electron withdrawing group;
- and
- at least one of R1, R2, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 or R14 is an electron donating group;
- and
- a reactive linking group linking the aryl carbon of the quencher moiety to L1, either directly or by way of a linker NR″R″ moiety, where each R″ is independently C1-C12 alkyl;
- L1, L3 and L4 are each, independently of one another, selected from a bond, C1-C12 alkyldiyl, C1-C12 alkoxyldiyl, C1-C12 alkylaminodiyl, C1-C12 alkylamidediyl, C5-C14 aryldiyl, and 1-20 ethyleneoxy units;
- L2 is a C1-C12 alkoxyldiyl, wherein the hydroxyl group is attached to a phosphodiester moiety of a first nucleotide;
- A is a cleavable linker;
- Y is selected from N and CR, where R is selected from H, C1-C6 alkyl, and C5-C14 aryl;
- and
- represents the solid support.
Dependencies are inferred from the claim text.