CA2333253A1 - Molecular torches - Google Patents
Molecular torches Download PDFInfo
- Publication number
- CA2333253A1 CA2333253A1 CA002333253A CA2333253A CA2333253A1 CA 2333253 A1 CA2333253 A1 CA 2333253A1 CA 002333253 A CA002333253 A CA 002333253A CA 2333253 A CA2333253 A CA 2333253A CA 2333253 A1 CA2333253 A1 CA 2333253A1
- Authority
- CA
- Canada
- Prior art keywords
- target
- torch
- binding domain
- domain
- closing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
Abstract
The present invention features "molecular torches" and the use of molecular torches for detecting the presence of a target nucleic acid sequence. Molecular torches contain a target binding domain, a target closing domain, and a joining region. The target binding domain is biased towards the target sequence such that the target binding domain forms a more stable hybrid with the target sequence than with the target closing domain under the same hybridization conditions. The joining region facilitates the formation or maintenance of a closed torch.
Claims (37)
1. A method for determining whether a target nucleic acid sequence is present in a sample comprising the steps of:
a) contacting said sample with a molecular torch comprising:
target detection means for hybridizing to said target sequence, if present, to produce an open torch, torch closing means for hybridizing to said target detecting means in the absence of said target sequence to produce a closed torch, and joining means for joining said target detection means and said torch closing means, wherein said joining means facilitates the formation of said closed torch in the absence of said target sequence; and b) detecting the presence of said open torch as an indication of the presence of said target sequence in said sample.
a) contacting said sample with a molecular torch comprising:
target detection means for hybridizing to said target sequence, if present, to produce an open torch, torch closing means for hybridizing to said target detecting means in the absence of said target sequence to produce a closed torch, and joining means for joining said target detection means and said torch closing means, wherein said joining means facilitates the formation of said closed torch in the absence of said target sequence; and b) detecting the presence of said open torch as an indication of the presence of said target sequence in said sample.
2. The method of claim 1, wherein said target sequence is generated using a transcription-associated amplification and said molecular torch is added to said sample prior to said amplification.
3. A method for determining whether a target nucleic acid sequence is present in a sample comprising the steps of:
a) contacting said sample with a molecular torch comprising:
a target binding domain, a target closing domain, and a joining region joining together said target binding and target closing domains, wherein said joining region facilitates the formation of a closed torch in the absence of said target sequence; and b) detecting the presence of said open torch as an indication of the presence of said target sequence in said sample.
a) contacting said sample with a molecular torch comprising:
a target binding domain, a target closing domain, and a joining region joining together said target binding and target closing domains, wherein said joining region facilitates the formation of a closed torch in the absence of said target sequence; and b) detecting the presence of said open torch as an indication of the presence of said target sequence in said sample.
4. The method of claim 3, wherein said target binding domain or said target closing domain comprises a label and said label produces a signal when a target binding domain:target closing domain hybrid is formed that is different than a signal produced when said target binding domain:target closing domain hybrid is not formed.
5. The method of claim 3, wherein said target binding domain comprises a first label and said target closing domain comprises a second label, and said first and second labels interact when said target binding domain and said target closing domain form a target binding domain:target closing domain hybrid to produce a signal that is different than when said target opening and target closing domains do not form said target binding domain:target closing domain hybrid, wherein said step b) detects an interaction change between said first and second labels as an indication of the presence of said open torch and said target sequence in said sample.
6. The method of claim 5, wherein said first and second labels are a luminescent/quencher pair.
7. The method of claim 5, wherein said first and second labels are a fluorophore/quencher pair.
8. The method of claim 5, wherein said first and second labels are a luminescent/adduct pair.
9. The method of claim 5, wherein said first and second labels are a Förrester energy transfer pair.
10. The method of claim 5, wherein said first and second labels are a dye dimer pair.
11. The method of claim 5, wherein said target sequence is generated using a transcription-associated amplification and said molecular torch is added to said sample prior to said amplification.
12. The method of claim 5, wherein said joining region consists of one or more groups independently selected from the group consisting of: a polynucleotide, a polysaccharide and a polypeptide.
13. The method of claim 5, wherein said joining region includes first and second polynucleotides, or derivatives thereof, said first and second polynucleotides, or derivatives thereof, being substantially complementary to one.
14. The method of claim 13, wherein said target sequence is RNA and said target binding domain is substantially comprised of independently selected 2'-methoxy or 2'-fluoro substituted ribonucleotides, and wherein said target closing domain is substantially comprised of independently selected deoxyribonucleotides.
15. The method of claim 13, wherein said target binding domain is substantially comprised of independently selected 2'-methoxy substituted ribonucleotides.
16. A method of determining whether a target nucleic acid sequence is present in a sample comprising the steps of:
a) contacting said sample with a molecular torch comprising, a target binding domain, a target closing domain, and a joining region joining together said target binding and target-closing domains, wherein said joining region facilitates the formation of a closed torch in the absence of said target sequence;
b) exposing a mixture comprising said sample and said torch to denaturing conditions such that said target binding domain and said target closing domain do not form a stable target binding domain:target closing domain hybrid;
c) exposing said mixture to hybridization conditions such that, in the absence of said target sequence, a target binding domain:target closing domain is formed, and in the presence of said target sequence a target binding domain:target sequence hybrid is formed in steps b) or c) to produce an open torch and said target binding domain:target sequence hybrid is more stable than said target binding domain:target closing domain hybrid; and d) detecting the presence of said open torch as an indication of the presence of said target sequence in said sample.
a) contacting said sample with a molecular torch comprising, a target binding domain, a target closing domain, and a joining region joining together said target binding and target-closing domains, wherein said joining region facilitates the formation of a closed torch in the absence of said target sequence;
b) exposing a mixture comprising said sample and said torch to denaturing conditions such that said target binding domain and said target closing domain do not form a stable target binding domain:target closing domain hybrid;
c) exposing said mixture to hybridization conditions such that, in the absence of said target sequence, a target binding domain:target closing domain is formed, and in the presence of said target sequence a target binding domain:target sequence hybrid is formed in steps b) or c) to produce an open torch and said target binding domain:target sequence hybrid is more stable than said target binding domain:target closing domain hybrid; and d) detecting the presence of said open torch as an indication of the presence of said target sequence in said sample.
17. A method of claim 16, where said step b) raises the temperature of said mixture, and said step c) lowers the temperature of said mixture.
18. The method of claim 17, wherein said target binding domain or said target closing domain comprises a label and said label produces a signal when said target binding domain:target closing domain hybrid is formed that is different than a signal produced when said target binding domain:target closing domain hybrid is not formed.
19. The method of claim 17, wherein said target binding domain comprises a first label and said target closing domain comprises a second label, and wherein said first and second labels interact when said target binding domain and said target closing domain form said target binding domain:target closing domain hybrid to produce a signal that is different than when said target opening and target closing domains do not form said target binding domain:target closing domain hybrid, wherein said step c) detects an interaction change between said first and second labels as an indication of the presence of said open torch and said target sequence in said sample.
20. The method of claim 19, wherein said first and second labels are a luminescent/quencher pair.
21. The method of claim 19, wherein said first and second labels are a fluorophore/quencher pair.
22. The method of claim 19, wherein said first and second labels are a luminescent/adduct pair.
23. The method of claim 19, wherein said first and second labels are a Förrester energy transfer pair.
24. The method of claim 19, wherein said first and second labels are a dye dimer pair.
25. The method of claim 16, wherein said target sequence is generated using a transcription-associated amplification and said molecular torch is added to said sample prior to said amplification.
26. The method of claim 16, wherein said joining region consists of one or more groups independently selected from the group consisting of: a polynucleotide, a polysaccharide and a polypeptide.
27. The method of claim 16, wherein said joining region includes first and second polynucleotides, or derivatives thereof, said first and second polynucleotides, or derivatives thereof, being substantially complementary to one.
28. The method of claim 16, wherein said target sequence is RNA and said target binding domain is substantially comprised of independently selected 2'-methoxy or 2'-fluoro substituted ribonucleotides, and wherein said target closing domain is substantially comprised of independently selected deoxyribonucleotides.
29. The method of claim 28, wherein said target binding domain is substantially comprised of independently selected 2'-methoxy substituted ribonucleotides.
30. A molecular torch comprising:
a target detection means for hybridizing to a target sequence to produce an open torch, a torch closing means for hybridizing to said target detecting means in the absence of said target sequence to produce a closed torch, and a joining means for facilitating the formation of said closed torch in the absence of said target sequence.
a target detection means for hybridizing to a target sequence to produce an open torch, a torch closing means for hybridizing to said target detecting means in the absence of said target sequence to produce a closed torch, and a joining means for facilitating the formation of said closed torch in the absence of said target sequence.
31. The molecular torch of claim 30, wherein said target detection means comprises a first label, said torch closing means comprises a second label, and said first and second labels interact when present in said open torch to produce a signal that is different than when present in said closed torch.
32. A molecular torch comprising:
a target binding domain, a target closing domain, wherein said target binding domain forms a more stable duplex with a perfect DNA or RNA complement of said target binding domain than with said target closing domain, and a joining region joining together said target opening and target closing domains, wherein said joining region facilitates the hybridization of said target binding domain to said target closing domain.
a target binding domain, a target closing domain, wherein said target binding domain forms a more stable duplex with a perfect DNA or RNA complement of said target binding domain than with said target closing domain, and a joining region joining together said target opening and target closing domains, wherein said joining region facilitates the hybridization of said target binding domain to said target closing domain.
33. The molecular torch of claim 32, wherein said target binding domain comprises a first label, said target closing domain comprises a second label, and said first and second labels interact when present in said open torch to produce a signal that is different than when present in said closed torch.
34. The torch of claim 33, wherein:
said target binding and target closing domains each comprise:
a backbone containing one or more groups independently selected from the group consisting of sugar-phosphodiester type linkages and peptide linkages, and nucleotide base recognition groups able to hydrogen bond to adenine, guanine, cytosine, thymine or uracil, joined to said backbone; and said joining region consists of one or more groups independently selected from the group consisting of: a nucleotide base recognition sequence, a polysaccharide, and a polypeptide.
said target binding and target closing domains each comprise:
a backbone containing one or more groups independently selected from the group consisting of sugar-phosphodiester type linkages and peptide linkages, and nucleotide base recognition groups able to hydrogen bond to adenine, guanine, cytosine, thymine or uracil, joined to said backbone; and said joining region consists of one or more groups independently selected from the group consisting of: a nucleotide base recognition sequence, a polysaccharide, and a polypeptide.
35. The torch of claim 34, wherein:
said target binding and target closing domains each comprise one or more groups independently selected from the group consisting of: a deoxyribonucleotide, a ribonucleotide, a 2'-methoxy substituted ribonucleotide, a 2'-fluoro substituted a ribonucleotide, and a peptide nucleic acid; and said joining region includes first and second polynucleotides, or derivatives thereof, said first and second polynucleotides, or derivatives thereof, being substantially complementary to one another.
said target binding and target closing domains each comprise one or more groups independently selected from the group consisting of: a deoxyribonucleotide, a ribonucleotide, a 2'-methoxy substituted ribonucleotide, a 2'-fluoro substituted a ribonucleotide, and a peptide nucleic acid; and said joining region includes first and second polynucleotides, or derivatives thereof, said first and second polynucleotides, or derivatives thereof, being substantially complementary to one another.
36. The torch of claim 35, wherein said target binding domain substantially comprises one or more groups independently selected from the group consisting of 2'-methoxy substituted ribonucleotides and 2'-fluoro substituted ribonucleotides, and wherein said target closing domain substantially comprises deoxyribonucleotides.
37. The torch of claim 36, wherein said first and second labels are a fluorophore/quencher pair.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US9161698P | 1998-07-02 | 1998-07-02 | |
| US60/091,616 | 1998-07-02 | ||
| PCT/US1999/015098 WO2000001850A2 (en) | 1998-07-02 | 1999-07-01 | Molecular torches |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2333253A1 true CA2333253A1 (en) | 2000-01-13 |
| CA2333253C CA2333253C (en) | 2010-09-07 |
Family
ID=22228747
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2333253A Expired - Lifetime CA2333253C (en) | 1998-07-02 | 1999-07-01 | Molecular torches |
Country Status (9)
| Country | Link |
|---|---|
| US (6) | US6361945B1 (en) |
| EP (1) | EP1092047B1 (en) |
| JP (2) | JP4646404B2 (en) |
| AT (1) | ATE440963T1 (en) |
| AU (1) | AU762728B2 (en) |
| CA (1) | CA2333253C (en) |
| DE (1) | DE69941333D1 (en) |
| ES (1) | ES2330812T3 (en) |
| WO (1) | WO2000001850A2 (en) |
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- 1999-07-01 WO PCT/US1999/015098 patent/WO2000001850A2/en active IP Right Grant
- 1999-07-01 DE DE69941333T patent/DE69941333D1/en not_active Expired - Lifetime
- 1999-07-01 CA CA2333253A patent/CA2333253C/en not_active Expired - Lifetime
- 1999-07-01 ES ES99932220T patent/ES2330812T3/en not_active Expired - Lifetime
- 1999-07-01 EP EP99932220A patent/EP1092047B1/en not_active Expired - Lifetime
- 1999-07-01 JP JP2000558240A patent/JP4646404B2/en not_active Expired - Fee Related
- 1999-07-01 AT AT99932220T patent/ATE440963T1/en not_active IP Right Cessation
- 1999-07-01 US US09/346,551 patent/US6361945B1/en not_active Expired - Lifetime
- 1999-07-01 AU AU48575/99A patent/AU762728B2/en not_active Expired
-
2001
- 2001-10-31 US US10/001,344 patent/US6534274B2/en not_active Expired - Lifetime
-
2003
- 2003-01-27 US US10/352,331 patent/US6835542B2/en not_active Expired - Lifetime
- 2003-01-27 US US10/352,355 patent/US6849412B2/en not_active Expired - Lifetime
-
2005
- 2005-01-27 US US11/044,098 patent/US7250259B2/en not_active Expired - Lifetime
- 2005-01-27 US US11/044,090 patent/US7252947B2/en not_active Expired - Fee Related
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2010
- 2010-08-23 JP JP2010186702A patent/JP2010252817A/en not_active Withdrawn
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| US20020090633A1 (en) | 2002-07-11 |
| WO2000001850A2 (en) | 2000-01-13 |
| US7252947B2 (en) | 2007-08-07 |
| US20030165957A1 (en) | 2003-09-04 |
| US6835542B2 (en) | 2004-12-28 |
| US6361945B1 (en) | 2002-03-26 |
| ES2330812T3 (en) | 2009-12-15 |
| US6849412B2 (en) | 2005-02-01 |
| ATE440963T1 (en) | 2009-09-15 |
| DE69941333D1 (en) | 2009-10-08 |
| US20030157542A1 (en) | 2003-08-21 |
| AU762728B2 (en) | 2003-07-03 |
| AU4857599A (en) | 2000-01-24 |
| EP1092047B1 (en) | 2009-08-26 |
| US7250259B2 (en) | 2007-07-31 |
| EP1092047A2 (en) | 2001-04-18 |
| CA2333253C (en) | 2010-09-07 |
| JP4646404B2 (en) | 2011-03-09 |
| WO2000001850A3 (en) | 2000-04-06 |
| JP2002519073A (en) | 2002-07-02 |
| US20050130216A1 (en) | 2005-06-16 |
| US20050123988A1 (en) | 2005-06-09 |
| JP2010252817A (en) | 2010-11-11 |
| US6534274B2 (en) | 2003-03-18 |
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