Monitoring oligonucleotide binding processes using chemiluminescence quenching
View Patent ↗Oligonucleotide building processes are monitored by means of an oligonucleotide probe which in one embodiment is labelled at one end with a chemiluminescent label and at the other end with a quencher molecule. The conformation of the oligonucleotide probe changes according to whether the probe hybridises with a substantially complementary nucleic acid sequence. In the non-hybridised state the chemiluminescent label is sufficiently close proximity to the quencher that the chemiluminescent emission is substantially attenuated, but in the hybridised state the separation is such that there is little or no attenuation. Particular probes and emitter/quencher pairs are disclosed.
1. A method for detecting and/or quantifying a target nucleic acid sequence in a sample comprising:
contacting said sample with an oligonucleotide sequence containing at least a probe sequence capable of binding to said target nucleic acid sequence, wherein said oligonucleotide sequence is labelled with each of at least one chemiluminescent molecule and at least one quencher molecule capable of attenuating chemiluminescence from said chemiluminescent molecule, the chemiluminescent and quencher molecules being arranged so that the interaction thereof changes according to whether the probe sequence is bound to said target nucleic acid sequence, such that in one of the bound and unbound states said chemiluminescence is substantially attenuated, and in the other thereof there is reduced or no attenuation of the chemiluminescence;
chemically activating the chemiluminescent molecule; and
detecting the chemiluminescence emission from said chemiluminescent molecule and comparing said chemiluminescence emission with that corresponding to the absence of said target nucleic acid sequence thereby detecting and/or quantifying said target nucleic acid sequence.
2. A method according to claim 1 , wherein, in the unbound state of the oligonucleotide sequence, the quencher molecule substantially quenches the chemiluminescence emission from said chemiluminescent molecule.
3. A method according to claim 1 or claim 2 , wherein:
(i) in the absence of the target nucleic acid sequence to be detected, the chemiluminescent and quencher molecules are held in sufficiently close proximity to each other, such that chemiluminescence quenching occurs, by a bridging molecule capable of binding to the opposite end regions of said probe sequence when said end regions are positioned adjacent or substantially adjacent to each other; and
(ii) in the presence of the target nucleic acid sequence to be detected, the chemiluminescent and quencher molecules are separated upon preferential binding of the probe sequence to the target nucleic acid sequence such that little or no chemiluminescence quenching occurs.
4. A method according to claim 3 , wherein said bridging molecule comprises a second oligonucleotide sequence which is complementary to groups of sequential nucleotides adjacent to the two ends of the probe sequence.
5. A method according to claim 1 or claim 2 , wherein:
(i) in the absence of the target nucleic acid sequence to be detected, the chemiluminescent and quencher molecules are held in sufficiently close proximity to each other due to their affinity for each other, such that chemiluminescence quenching occurs; and
(ii) in the presence of the target nucleic acid sequence to be detected, the chemiluminescent and quencher molecules are separated upon preferential binding of the probe sequence to the target nucleic acid sequence such that little or no chemiluminescence quenching occurs.
6. A method according to claim 5 , wherein:
(i) in the absence of the target nucleic acid sequence to be detected, the chemiluminescent and quencher molecules are held in sufficiently close proximity to each other, such that chemiluminescence quenching occurs, by the presence of flanking oligonucleotide sequences which are complementary to each other adjacent to the two ends of the probe sequence such that intra-molecular binding occurs; and
(ii) in the presence of the target nucleic acid sequence to be detected, the chemiluminescent and quencher molecules are separated upon preferential binding of the probe sequence to the target nucleic acid sequence such that little or no chemiluminescence quenching occurs.
7. A method according to claim 5 , wherein:
(i) in the absence of the target nucleic acid sequence to be detected, the chemiluminescent and quencher molecules are held in sufficiently close proximity to each other, such that chemiluminescence quenching occurs, by the presence of pairs of chemical species having affinity for each other and which are positioned in or comprise any of the regions of the oligonucleotide sequence comprising the following groups: probe sequence, flanking sequence, linker groups, chemiluminescent emitter, chemiluminescent quencher; and
(ii) in the presence of the target nucleic acid sequence to be detected the chemiluminescent and quencher molecules are separated upon preferential binding of the probe sequence to the target nucleic acid sequence such that little or no chemiluminescence quenching occurs.
8. A method for detecting and/or quantifying a target nucleic acid sequence in a sample comprising:
contacting said sample with at least two oligonucleotides having different sequences capable of simultaneously binding to said target nucleic acid sequence, wherein said at least two oligonucleotides are labelled respectively with at least one chemiluminescent molecule and at least one quencher molecule capable of attenuating chemiluminescence when said two labelled oligonucleotides bind to said target nucleic acid sequence substantially adjacent to each other;
chemically activating the chemiluminescent molecule; and
detecting the chemiluminescence emission from said chemiluminescent molecule and comparing said chemiluminescence emission with that corresponding to the absence of said target nucleic acid sequence thereby detecting and/or quantifying said target nucleic acid sequence.
9. A method for detecting and/or quantifying a target nucleic acid sequence in a sample comprising:
contacting said sample with at least two oligonucleotides capable of binding to each other, wherein said at least two oligonucleotides are labelled respectively with at least one chemiluminescent molecule and at least one quencher molecule capable of attenuating chemiluminescence when said labelled oligonucleotides are bound to each other, and wherein there is little or no attenuation of said chemiluminescence from the chemiluminescent molecule when the binding of said labelled oligonucleotides to each other is inhibited by the presence of said target nucleic acid sequence;
chemically activating the chemiluminescent molecule; and
detecting the chemiluminescence emission from said chemiluminescent molecule and comparing said chemiluminescence emission obtained in the presence and absence of said target nucleic acid sequence thereby detecting and/or quantifying said target nucleic acid sequence.
10. A method according to claim 1 or claim 2 , wherein at least two different target nucleic acid sequences are detected and/or quantified by use of at least two mutually distinguishable oligonucleotide systems having different sequences, each being labelled with a different chemiluminescent molecule and the same or a different quencher molecule.
11. A method according to claim 8 or 9 , wherein at least two different target nucleic acid sequences are detected and/or quantified by use of at least two mutually distinguishable oligonucleotide systems, each having a different sequence capable of hybridising to a respective one of said target nucleic acid sequences and each labelled with a different chemiluminescent molecule, and at least one oligonucleotide having a different sequence capable of hybridising to at least one of said target nucleic acid sequences and labelled with a quencher molecule.
12. A method according to claim 11 , wherein at least two different target nucleic acid sequences are detected and/or quantified by use of at least two oligonucleotides having different sequences each labelled with different chemiluminescent molecules and at least two oligonucleotides having different sequences and labelled with different quencher molecules.
13. A method according to claim 1 or claim 2 , wherein the chemiluminescent molecules are selected from the group of compounds of which the following form a structural basis: acridine, phenanthridine, benzacridine, quinoline, and pyridine.
14. A method according to claim 13 , wherein the chemiluminescent molecule is an acridinium salt.
15. A method according claim 13 , wherein the chemiluminescent molecule is an acridan.
16. A method according to claim 1 or claim 2 , wherein the chemiluminescent molecule emits light within the wavelength range 200–700 nm.
17. A method according to claim 1 or claim 2 , wherein the quencher molecule absorbs light within the wavelength range 200–700 nm.
18. A method according to claim 1 or claim 2 , wherein the quencher molecule is an azo dye.
19. A method according to claim 1 or claim 2 , wherein the quencher molecule is a derivative of methyl red.
20. A labelled oligonucleotide for detecting and/or quantifying a target nucleic acid sequence comprising an oligonucleotide probe sequence capable of binding to said target nucleic acid sequence, said oligonucleotide being labelled with a chemiluminescent molecule requiring chemical activation and a quencher molecule and being arranged such that the quencher molecule and the chemiluminescent molecule requiring chemical activation interact to cause a change in a subsequent chemiluminescent emission according to whether the labelled oligonucleotide is bound to said target nucleic acid sequence.
21. A labelled oligonucleotide according to claim 20 , wherein, when not bound to said target nucleic acid sequence, said quencher molecule causes quenching of said chemiluminescence, but binding to said target nucleic acid sequence prevents or reduces said quenching.
22. A labelled oligonucleotide according to claim 20 or claim 21 , wherein said oligonucleotide is between 15 and 45 nucleotide.
23. A labelled oligonucleotide according to claim 20 or claim 21 , wherein said chemiluminescent molecule is an acridinium salt.
24. A labelled oligonucleotide according to claim 20 or claim 21 , wherein said quencher molecule is an azo dye.
25. A labelled oligonucleotide according to claim 24 , wherein said quencher molecule is a derivative of methyl red.
26. A kit comprising a labelled oligonucleotide according to claim 20 or claim 21 and reagent means for initiating said chemiluminescent reaction.