Nanoprobes for detection or modification of molecules

• US 20100227913A1
• Filed: 12/12/2006
• Published: 09/09/2010
• Est. Priority Date: 12/12/2005
• Status: Active Grant

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: National Aeronautics and Space Administration (Government of the United States of America)
• Inventors: Needle, Danielle, Lyakhov, Ilya G., Schneider, Thomas D.
• Granted Patent: US 8,344,121 B2
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/30 Phosphoric diester hydrolysing, i.e. nuclease
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2535/107 Maxam and Gilbert method, i.e. sequential release and detection of nucleotides
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The disclosure provides probes for one or more target molecules. In particular examples, the probes include a molecular linker and first and second functional groups linked and spaced by the molecular linker, wherein the functional groups are capable of interacting with one another or with the target biomolecule in a predetermined reaction, and wherein the molecular linker maintains the first and second functional groups sufficiently spaced from one another such that the functional groups do not substantially interact in an absence of the target biomolecule. In the presence of the target biomolecule the functional groups interact (with each other, with the target biomolecule, or both), and in some examples a detectable signal is produced. In some examples, the functional groups can detect or modify a target molecule. Also provided are methods of using the probes, for example to detect or modify a target molecule.

Claims

1. A probe for a target biomolecule, comprising:

a molecular linker; and

first and second functional groups linked and spaced by the molecular linker, wherein the functional groups are capable of interacting with one another or with the target biomolecule in a predetermined reaction, wherein the molecular linker links the first and second functional groups sufficiently spaced from one another such that the functional groups do not substantially interact in an absence of the target biomolecule but interact in a presence of the target biomolecule to provide a signal of the presence of the biomolecule.

2. The probe of claim 1, wherein the molecular linker maintains the first and second functional groups sufficiently spaced a distance from one another to avoid substantial entanglement of the first and second functional groups in an absence of the target biomolecule.

3. The probe of claim 1, wherein at least a portion of the molecular linker is of a sufficient rigidity to reduce interaction of the first and second functional groups in the absence of the target biomolecule.

4. The probe of claim 3, wherein at least a portion of the molecular linker comprises a molecular rod having a persistence length at least as great as a persistence length of a double-stranded DNA (dsDNA) of at least 10-150 nucleotides.

5. The probe of claim 1, wherein the molecular linker is of a sufficient length to substantially avoid interaction of the first and second functional groups in the absence of the target biomolecule, and allow interaction of the first and second functional groups in the presence of the target biomolecule.

6. The probe of claim 1, wherein the molecular linker comprises a tether, a molecular rod, or combinations thereof

7. The probe of claim 3, wherein the molecular linker of sufficient rigidity comprises a molecular rod.

8. The probe of claim 7, wherein the molecular linker of sufficient rigidity comprises multiple molecular rods linked by tethers.

9. The probe of claim 7, wherein the molecular linker of sufficient rigidity comprises multiple tethers linked by molecular rods.

10. The probe of claim 6, wherein the tether comprises polyethylene glycol (PEG).

11. The probe of claim 10, wherein the tether consists of PEG.

12. The probe of claim 6, wherein the tether is less than 187 Å

in length.

13. The probe of claim 2, wherein the molecular rod comprises a double-stranded DNA (dsDNA) molecule of at least 10 nucleotides.

14. The probe of claim 13, wherein the dsDNA molecule comprises 10-140 nucleotides.

15. The probe of claim 1, wherein the functional groups permit detection or modification of the target biomolecule.

16. The probe of claim 1, wherein the functional groups comprise a nucleic acid molecule, a protein detection agent, a protein, a label, or combinations thereof.

17. The probe of claim 16, wherein the protein detection agent comprises an antibody that specifically binds to a target protein.

18. The probe of claim 16, wherein the nucleic acid molecule comprises an antisense molecule that specifically hybridizes to a target nucleic acid sequence.

19. The probe of claim 17, wherein the first functional group comprises an antibody that can specifically bind to the target protein, and the second functional group comprises one or more DNA binding sites that can specifically bind to the target protein.

20. The probe of claim 19, wherein the target protein comprises a DNA binding protein.

21. The probe of claim 16, wherein the first and second functional groups comprise antibodies that can specifically bind to a target protein.

22. The probe of claim 18, wherein the first functional group comprises a first antisense oligonucleotide, and wherein the second functional group comprises a second antisense oligonucleotide, wherein the first and second antisense oligonucleotides can hybridize to a target nucleic acid sequence under high stringency conditions.

23. The probe of claim 16, wherein the first functional group comprises a nucleic acid sequence capable of specifically hybridizing to the target nucleic acid, thereby forming a nucleic acid complex, and wherein the second functional group comprises a protein capable of cleaving the nucleic acid complex.

24. The probe of claim 23, wherein the nucleic acid sequence comprises an antisense DNA sequence that can hybridize to a target RNA under highly stringent conditions, thereby forming a DNA/RNA nucleic acid complex and wherein the protein comprises an RNase H that can cleave RNA in the DNA/RNA nucleic acid complex.

25. The probe of claim 16, wherein the first functional group comprises one or more DNA binding sites that can specifically bind to a target protein, and wherein the second functional group comprises a protein capable of cleaving the protein.

26. The probe of claim 16, wherein the first functional group comprises a polymerase, and wherein the second functional group comprises a non-hydrolyzable dNTP.

27. The probe of claim 26, further comprising multiple functional groups linked and spaced by the molecular linker to the polymerase, wherein the multiple functional groups comprise non-hydrolyzable dNTPs.

28. The probe of claim 16, wherein the functional group comprises a label.

29. The probe of claim 28, wherein the first functional group comprises an acceptor fluorophore and the second functional group comprises a donor fluorophore.

30. A method of detecting a target biomolecule, comprising:

contacting a sample with the probe of claim 1 under conditions sufficient for the functional groups to specifically interact with the target biomolecule, wherein interaction of the functional groups results in the production of a signal by a label on the probe;

detecting the signal when the probe interacts with target biomolecule.

31. The method of claim 30, wherein the label comprises an acceptor fluorophore and wherein when interaction of the functional groups brings the acceptor fluorophore into a proximity with a donor fluorophore to permit excitation of the acceptor fluorophore by the donor fluorophore.

32. The method of claim 31, wherein detecting a signal comprises detecting a fluorescent signal emitted from the acceptor fluorophore.

33. The method of claim 30, wherein the sample comprises a cell, and wherein contacting the sample with the probe comprises contacting the cell with the probe under conditions sufficient for the probe to enter the cell.

34. The method of claim 33, wherein the sample is obtained from a subject, and the method is performed ex vivo.

35. The method of claim 30, wherein contacting the sample with the probe comprises administration of the composition to a subject.

36. The method of claim 30, wherein the target biomolecule comprises a protein or a nucleic acid sequence.

37. The method of claim 36, wherein the protein comprises one or more mutations associated with disease.

38. A method of modifying a target biomolecule, comprising:

exposing a sample to the composition of claim 21 under conditions sufficient for the nucleic acid sequence to bind to the target biomolecule, and sufficient for the protein to cleave the target biomolecule.

39. The method of claim 38, further comprising detecting the cleaving of the target biomolecule.

40. A method of treating a subject, comprising:

administering a therapeutic amount of the composition of claim 23 or 25 to a subject, wherein the subject has a disorder that would benefit from the cleavage of the target biomolecule.

41. A method of sequencing a sample nucleic acid sequence, comprising:

contacting the sample nucleic acid sequence to an oligonucleotide primer and the probe of claim 27 in the presence of a mixture of non-labeled hydrolyzable nucleotides, wherein each non-hydrolyzable dNTP includes a label that emits a signal corresponding to the particular nucleotide complementary to a nucleotide on the sample nucleic acid sequence;

detecting the signal as each non-hydrolyzable dNTP is exposed to the sample nucleic acid molecule; and

allowing one of the non-labeled hydrolyzable nucleotides to be incorporated into a synthesized nucleic acid molecule complementary to the sample nucleic acid molecule.