Tunable fluorescence using cleavable linkers

Patent metadata

Field	Value
Publication number	US-9845493-B2
Application number	US-201414784939-A
Country	US
Kind code	B2
Filing date	Apr 21, 2014
Priority date	Apr 19, 2013
Publication date	Dec 19, 2017
Grant date	Dec 19, 2017

Abstract

The invention relates to cleavable chemistry in general, and in particular, to tunable fluorescence using cleavable linkers present in fluorochrome-quencher conjugates.

First claim

The invention claimed is:

1. Fluorochrome-quencher conjugate having the structure
F-L1-B-L2-Q
wherein
F is at least one fluorochrome;
Q is at least one quenching molecule for the at least one fluorochrome, or is another fluorochrome F, or is another fluorochrome other than F;
L 1 is at least one first cleavable linker molecule;
L 2 is at least one second cleavable linker molecule, wherein said L 1 and L 2 are different so that fluorophores can be selectively turned on upon specific cleavage of said L 1 and L 2 ; and
B is a linking moiety or a target molecule of interest;
wherein L 2 is directly attached to Q via amide coupling to form part of a modular system of the conjugate.

2. The conjugate of claim 1 , wherein each of the at least one first cleavable linker and the at least one second cleavable linker is independently selected from the group consisting of azido linker, allyl linker, disulfide linker, phosphorothioate linker, diol linker, diazobenzene linker, acylsulfonamide linker, and silyl linker.

3. The conjugate of claim 2 , wherein the at least one first and/or the at least one second cleavable linker are independently selected from the group consisting of:

wherein x is independently selected and is an integer of between 1 and 10.

4. The conjugate of claim 1 , wherein the at least one fluorochrome is a fluorophore selected from the group consisting of Cy3, Cy5, FAM, TET, HEX, TMR, ROX, Texas red, LC red 640, and LC red 70.

5. The conjugate of claim 1 , wherein the at least one quenching molecule is selected from the group consisting of QSY-7, DDQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, BHQ-2, DDQ-II, Iowa Black RQ, QSY-21, and BHQ-3.

6. The conjugate of claim 1 , wherein Q is another fluorochrome other than F, wherein Q undergoes Forster resonance energy transfer (FRET) with F such that a fluorescence emission wavelength change occurs upon removal of Q.

7. The conjugate of claim 6 , wherein the another fluorochrome other than F is Cy3 and Q is Cy5.

8. The conjugate of claim 5 , wherein F is Cy3 and Q is QSY-7.

9. The conjugate of claim 1 , wherein F, B, L 1 and L 2 are coupled to each other through conjugation chemistry.

10. The conjugate of claim 9 , wherein F, B, L 1 and L 2 are coupled to each other by amide chemistry to form the modular system of the conjugate.

11. The conjugate of claim 1 , wherein L 1 -F and L 2 -Q are separately conjugated to a respective biomolecule and are spaced at a distance apart for quenching of the fluorescence or FRET to occur.

12. The conjugate of claim 11 , wherein the biomolecule is selected from the group consisting of nucleic acids, lipids, peptides and proteins.

13. The conjugate of claim 1 , wherein B is a linking moiety and further comprises a reactive group R for coupling the conjugate to a target molecule.

14. The conjugate of claim 13 , wherein B has the structure:

wherein R is selected from the group consisting of hydroxyl, carboxyl, and amine; and
wherein x is independently selected and is an integer of between 1 and 6.

15. The conjugate of claim 1 , wherein the target molecule is an amino acid, a peptide, a polypeptide, a nucleotide, an oligonucleotide, or a polynucleotide.

16. The conjugate of claim 1 , wherein the conjugate is

17. A method of detecting a target molecule, comprising:
contacting a sample suspected of containing the target molecule with a fluorochrome-quencher conjugate according to claim 1 ; and
measuring the fluorescence.

18. The method of claim 17 , further comprising selectively cleaving the at least one first and/or the at least one second linker molecule prior to measuring the fluorescence, wherein cleavage is achieved by adding at least one selective cleavage reagent or incubating the sample under conditions that allow selective cleavage of the at least one first or the at least one second linker molecule.

19. A method of detecting two or more targets of interest, comprising:
contacting a sample suspected of containing the two or more targets of interest with two or more fluorochrome-quencher conjugates according to claim 1 , wherein F and Q of each of the two or more fluorochrome-quencher conjugate are the same, wherein L 1 and L 2 of each of the two or more fluorochrome-quencher conjugate are different, and L 2 is directly attached to Q via amide coupling; and
measuring the fluorescence.

20. The method of claim 19 , further comprising selectively cleaving the at least one first and/or the at least one second linker molecule in each of the two or more fluorochrome-quencher conjugate prior to measuring the fluorescence, wherein cleavage is achieved by adding at least one selective cleavage reagent or incubating the sample under conditions that allow selective cleavage of the at least one first or the at least one second linker molecule of each of the two or more fluorochrome-quencher conjugate.

21. The conjugate of claim 1 , wherein the at least one quenching molecule is Dabcyl.

22. The method of claim 17 , wherein the fluorochrome-quencher comprises Dabcyl.

23. The method of claim 19 , wherein Q is Dabcyl.

Assignees

• Agency Science Tech & Res

Inventors

• Brenner Sydney
• Teo Yin Nah
• Tan Pee Mei

Classifications

• C07F9/2408

  of hydroxyalkyl compounds · CPC title

• C12Q1/6818 Primary

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C07D207/46

  with hetero atoms directly attached to the ring nitrogen atom · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• C09B11/08

  Phthaleins; {Phenolphthaleins; Fluorescein} · CPC title