CAPTURE OF TARGET DNA AND RNA BY PROBES COMPRISING INTERCALATOR MOLECULES

• US 20130230856A1
• Filed: 10/27/2011
• Published: 09/05/2013
• Est. Priority Date: 10/27/2010
• Status: Abandoned Application

• Current Assignee: Quantibact A/S
• Original Assignee: Quantibact A/S
• Inventors: Schneider, Uffe Vest, Johnk, Nina, Lisby, Jan Gorm
• Application Number:
  US13/881,714
• Publication Number:
  US 20130230856A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to a technology for specific capture of single stranded target Polynucleotide by a complementary probe comprising one or more intercalator molecules. The method further involves removal of one or more types of bases in the single stranded target Polynucleotide prior to interaction with the complementary probe. This results in generation of one or more abasic sites which can interact with and/or into where the intercalator molecule can be inserted.

Claims

1. A method for capturing a target polynucleotide from a sample obtained from biological material, the method comprising the steps of:

(i) providing a double stranded target polynucleotide;

(ii) destabilizing said double stranded target polynucleotide by removing one or more bases, such as one or more of the nucleobases A, T, U, C or G, 5-hydroxymethyl-dC, 5-methylcytosine (m⁵C), pseudouridine (Ψ

), dihydrouridine (D), inosine (I), 7-methylguanosine (m⁷G), hypoxanthine, xanthine and their 2′

-O-Methyl-derivatives and/or N-Methyl-derivatives from said target polynucleotide, thereby generating one or more abasic sites and

(iii) denaturing said destabilized double stranded target polynucleotide to generate single stranded target polynucleotide, and

(iv) capturing said single stranded target polynucleotide with a complementary oligonucleotide probe having a length of 15 to 35 nucleotides, wherein the complementary oligonucleotide probe comprises one or more intercalator molecules inserted into the backbone-structure of said oligonucleotide probe, wherein said one or more intercalator molecule(s) fit morphologically into the one or more abasic sites of the complementary polynucleotide target sequence.

2. The method according to preceding claim 1 wherein the complementary oligonucleotide probe comprises naturally occurring nucleotides and/or nucleotides which are not known to occur in nature such as those selected from the group consisting of RNA, α

-L-RNA, β

-D-RNA, 2′

-R-RNA, DNA, LNA, PNA, PMO, TNA, GNA, oligonucleotide N3′

→

P5′

phosphoramidates, BNA, α

-L-LNA, HNA, MNA, ANA, CAN, INA, CeNA, (2′

-NH)-TNA, (3′

—

NH)-TNA, α

-L-Ribo-LNA, α

-L-Xylo-LNA, β

-D-Ribo-LNA, β

-D-Xylo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, α

-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β

-D-Ribopyranosyl-NA, α

-L-Lyxopyranosyl-NA, 2′

-OR-RNA, 2′

-AE-RNA, and combinations and modifications thereof.

3. The method according to claim 1, wherein the method further comprises one or more washing steps in order to remove unbound nucleotide material.

4. The method according to claim 1, wherein the one or more abasic sites are 2 or more abasic sites, such as 3 or more abasic sites.

5. The method according to claim 1, wherein the method further comprises conversion of one or more types of bases in the double stranded target polynucleotide to another chemical entity.

6. The method according to claim 5, wherein the method further comprises destabilisation of said double stranded target polynucleotide by removal of one or more chemical entities from said double stranded target polynucleotide.

7. The method according to claim 1, wherein the method further comprises conversion of one or more C'"'"'s in the target polynucleotide to one or more U'"'"'s.

8. The method according to claim 7, wherein the conversion of one or more C'"'"'s in the target polynucleotide to one or more U'"'"'s is preformed by bisulphite treatment.

9. The method according to claim 1, wherein A is removed from said double stranded target polynucleotide and/or single stranded polynucleotide.

10. The method according to claim 1, wherein T is removed from said double stranded target polynucleotide and/or single stranded polynucleotide.

11. The method according to claim 1, wherein C is removed from said double stranded target polynucleotide and/or single stranded Polynucleotide.

12. The method according to claim 1, wherein G is removed from said double stranded target polynucleotide and/or single stranded Polynucleotide.

13. The method according to claim 1, wherein U is removed from said double stranded target Polynucleotide and/or single stranded Polynucleotide.

14. The method according to claim 1, wherein the removal is performed by one or more enzymes and/or physical stress and/or temperature change.

15. The method according to claim 13, wherein the removal of U is performed by use of uracil dehydrogenase.

16. The method according to claim 9, wherein the removal of A is performed by adjustment of the pH.

17. The method according to claim 1, wherein 1, 2, or 3 types of the bases from the target polynucleotide is/are removed.

18. The method according to claim 1, wherein the total number of bases that are removed from the target Polynucleotide can be selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and more than 20 bases.

19. The method according to claim 1, wherein the total number of intercalator molecules can be selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and more than 20 intercalator molecules.

20. The method according to claim 1, wherein an intercalator molecule has been inserted into from 10% to 100% of the abasic sites in the target DNA and/or RNA such as from 10% to 20%, for example from 20% to 30%, such as from 30% to 40%, for example from 40% to 50%, such as from 50% to 60%, for example from 60% to 70%, such as from 70% to 80%, for example from 80% to 90%, such as from 90% to 100%, or any combination thereof.

21. The method according to claim 1, wherein an intercalator molecule has been inserted into more than 10% of the abasic sites in the target DNA and/or RNA, such as more than 20%, for example more than 30%, such as more than 40%, for example more than 50%, such as more than 60%, for example more than 70%, such as more than 80%, for example more than 90%, such as more than 95%, for example 100%.

22. The method according to claim 1, wherein the insertion of the intercalator molecules results in increased melting temperature of the polynucleotide duplex consisting of the target Polynucleotide and the complementary probe.

23. The method according to claim 1, wherein the ratio between the number of intercalator molecules and the total number of bases in the complementary probe is from 1:

50 to 1;

2 such as from 1;

50 to 1;

40, for example 1;

40 to 1;

30, such as from 1;

30 to 1;

20, for example 1;

20 to 1;

10, such as from 1;

10 to 1;

5, for example 1;

5 to 1;

2, or any combination of these intervals.

24. The method according to claim 1, wherein the one or more intercalator molecules can be selected from the group consisting of TINA, INA, ortho-TINA, para-TINA, and AMANY.

25. The method according claim 1, wherein the size of the intercalator molecule is between 20 and 400 Å

, such as from 20-40 Å

, for example from 40-60 Å

, such as from 60-80 Å

, for example from 80-100 Å

, such as from 100-120 Å

, for example from 120-140 Å

, such as from 140-160 Å

, for example from 160-180 Å

, such as from 180-200 Å

, for example from 200-220 Å

, such as from 220-240 Å

, for example from 240-260 Å

, such as from 260-280 Å

, for example from 280-300 Å

, such as from 300-320 Å

, for example from 320-340 Å

, such as from 340-360 Å

, for example from 360-380 Å

, such as from 380-400 Å

, or any combination of these intervals.

26. The method according to claim 1, wherein the complementary probe comprises more than one type of intercalator molecules such as 2, 3, 4, 5 or more than 5 different types of intercalator molecules.

27. The method according to claim 1, wherein the complementary probe is connected to a support.

28. The method according to claim 27, wherein the support is selected from the group consisting of Poly(ether ether ketone) (PEEK), PP (polypropylene), PE (polyethylene), Poly(ethylene terephthalate) (PET), Poly(vinyl chloride) (PVC), Polyamide/nylon (PA), Polycarbonate (PC), Cyclic olefin copolymer (COC), Filter paper, Cotton, Cellulose, Poly(4-vinylbenzyl chloride) (PVBC), Poly(vinylidene fluoride) (PVDF), Polystyrene (PS), Toyopearl®

, Hydrogels, Polyimide (PI), 1,2-Polybutadiene (PB), LSR (Liquid silicon rubber), poly(dimethylsiloxane) (PDMS), fluoropolymers-and copolymers (e.g. poly(tetrafluoroethylene) (PTFE), Perfluoroethylene propylene copolymer (FEP), Ethylene tetrafluoroethylene (ETFE)), poly(methyl methacrylate) (PMMA), Nanoporous materials, Membranes, Mesostructured cellular foam (MCF), and singlewall or multiwall Carbon Nanotubes (SWCNT, MWCNT), particulate matters, beads, magnetic beads, non-magnetic beads, polystyrene beads, magnetic polystyrene beads, sepharose beads, sephacryl beads, polystyrene beads, agarose beads, polysaccharide beads, and polycarbamate beads.

29. The method according to claim 27, wherein the support is a solid support.

30. The method according to claim 29, wherein the solid support is selected from the group consisting of microtiter plate or other plate formats, reagent tubes, glass slides or other supports for use in array or microarray analysis, tubings or channels of micro fluidic chambers or devices and Biacore chips.

32. The method according to claim 1, wherein the complementary probe comprises one or more labels.

33. The method according to claim 32, wherein the one or more labels is selected from the group consisting of biotin, a fluorescent label, 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine, dyes, Cy2, Cy3, and Cy5, PerCP, phycobiliproteins, R-phycoerythrin (RPE), allophycoerythrin (APC), Texas Red, Princeston Red, Green fluorescent protein (GFP) and analogues thereof, conjugates of R-phycoerythrin or allophycoerythrin, inorganic fluorescent labels based on semiconductor nanocrystals (like quantum dot and Qdot™

nanocrystals), time-resolved fluorescent labels based on lanthanides like Eu3+ and Sm3+, haptens, DNP, digoxiginin, enzymic labels, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β

-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase and glucose oxidase (GO), luminiscence labels, luminol, isoluminol, acridinium esters, 1,2-dioxetanes, pyridopyridazines, radioactivity labels, isotopes of iodide, isotopes of cobalt, isotopes of elenium, isotopes of tritium, and isotopes of phosphor.

34. The method according to claim 33, wherein the biotin is detected by use of streptavidin-R-phycoerythrine.

35. The method according to claim 32, wherein the method further comprises a washing step prior to and/or after addition of the detection probe.

36. The method according to claim 32, wherein complementary detection probe comprises one or more intercalator molecules.

37. The method according to claim 36, wherein the total number of intercalator molecules can be selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and more than 20 different or identical intercalator molecules.

38. The method according to claim 1, wherein the target polynucleotide is derived from a human being, an animal, bacteria, virus, fungi, protozoa and/or plant.

39. The method according to claim 1, wherein the target polynucleotide is isolate from a sample from a human or animal body.

40. The method according to claim 1, wherein the target polynucleotide is isolate from humans, animals, birds, insects, plants, algae, fungi'"'"'s, yeast, viruses, bacteria and phages, multi-cellular and mono-cellular organisms.

41. The method according to claim 1, wherein the target polynucleotide is isolate from faeces, blood, semen, cerebrospinal fluid, sputum, vaginal fluid, urine, saliva, hair, other bodily fluids, tissue samples, whole organs, sweat, tears, skin cells, hair, bone, teeth or appropriate fluid or tissue from personal items (e.g. toothbrush, razor, etc.) or from samples (e.g. sperm or biopsy tissue or liquid) or other sub-structures of humans or animals.

42. The method according to claim 1, wherein the total number of different target polynucleotide sequences captured are selected from the group consisting of 1, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 95-100, 100-150, 150-200, 200-300, 300-500, 500-1000 and more than 1000 different, or any combination of these intervals.

43. The method according to claim 1 for diagnosing a genetic disease, wherein the genetic disease is associated with the target polynucleotide.

45. The method according to claim 43 wherein the disease to be diagnosed is selected from the group consisting of CADASIL syndrome;

Carboxylase Deficiency, Multiple, Late-Onset;

Cerebelloretinal Angiomatosis, familial;

Crohn'"'"'s disease, fibrostenosing;

Deficiency disease, Phenylalanine Hydroxylase;

Fabry disease;

Hereditary coproporphyria;

Incontinentia pigmenti;

Microcephaly;

Polycystic kidney disease;

Siderius X-linked mental retardation syndrome caused by mutations in the PHF8 gene and achondroplasia.

46. The method according to claim 43, wherein the disease to be diagnosed is cancer.

51. The method according to claim 1, wherein the sample is a feed, soil, food or drinking water.

31. (canceled)

44. (canceled)

47-50. -50. (canceled)

52-54. -54. (canceled)

55. An oligonucleotide probe consisting of 15 to 35 nucleotides, wherein the oligonucleotide probe comprises at least 2 intercalator molecules which fit morphologically into abasic sites of a complementary polynucleotide target, and wherein the nucleotides adjacent to the intercalator molecules of the oligonucleotide probe are complementary to the polynucleotide target.

56-79. -79. (canceled)

80. A complex comprising an oligonucleotide probe and a complementary polynucleotide target comprising at least two abasic sites,

wherein the oligonucleotide probe has a length of 15 to 35 nucleotides and comprises at least two intercalator molecules inserted into the backbone-structure of said oligonucleotide probe, and wherein said at least two intercalator molecules fit morphologically into the abasic sites of the complementary polynucleotide target.

81-104. -104. (canceled)