Compositions comprising oriented, immobilized macromolecules and methods for their preparation
View Patent ↗The present invention provides methods and compositions that facilitate the identification of structures of a variety of macromolecules. For instance, the present invention provides methods for the selective immobilization of macromolecules in an extended or oriented state. The present invention also provides compositions comprising macromolecules selectively immobilized in extended or oriented states.
1. A method for extending and selectively immobilizing a polynucleotide complex on a substrate comprising steps of:
(a) providing a first nucleic acid molecule comprising:
(i) a first target-specific sequence capable of binding to a target nucleic acid,
(ii) a first label attachment region, which is non-overlapping with the first target-specific sequence, comprising a first DNA sequence hybridized to a first RNA molecule that is attached to one or more detectable labels that emit light which constitutes a first signal,
(iii) a second label attachment region, which is non-overlapping with the first target-specific sequence and the first label attachment region, comprising a second DNA sequence hybridized to a second RNA molecule that is attached to one or more detectable labels that emit light which constitutes a second signal, and
(iv) a first moiety that is capable of selectively binding to the substrate;
(b) providing a second nucleic acid molecule comprising:
(i) a second target-specific sequence capable of binding to the target nucleic acid; wherein the first target-specific sequence and the second target-specific sequence bind to different regions of the target nucleic acid and
(ii) a second moiety that is capable of selectively binding to the substrate;
(c) contacting the first nucleic acid molecule and the second nucleic acid molecule with a sample containing a target nucleic acid under conditions sufficient for hybridization of the first and the second nucleic acid molecules to the target nucleic acid, thereby producing the polynucleotide complex;
(d) selectively binding the first moiety to the substrate, thereby selectively immobilizing the first nucleic acid of the polynucleotide complex to the substrate;
(e) applying to the polynucleotide complex a force sufficient to extend the polynucleotide complex such that the first signal and the second signal are spatially separated;
(f) selectively binding the second moiety to the substrate, thereby selectively immobilizing the second nucleic acid of the polynucleotide complex to the substrate and thereby selectively immobilizing the extended polynucleotide complex to the substrate; and
(g) removing the force, with the polynucleotide complex remaining selectively immobilized in an extended state.
2. The method of claim 1 , wherein the first nucleic acid molecule comprises additional label attachment regions which are non-overlapping with other label attachment regions.
3. The method of claim 2 , wherein at least one of the additional label attachment regions comprises a DNA sequence hybridized to an RNA molecule that is attached to one or more detectable labels that emit light.
4. The method of claim 2 , wherein at least one of the additional label attachment regions comprises a DNA sequence hybridized to an RNA molecule that is not attached to a detectable label that emits light.
5. The method of claim 2 , wherein at least one of the additional label attachment regions comprises a DNA sequence hybridized to an RNA molecule that is attached to one or more detectable labels that emit light and at least another one of the additional label attachment regions comprises a DNA sequence hybridized to an RNA molecule that is not attached to a detectable label that emits light.
6. The method of claim 1 , wherein the first nucleic acid molecule further comprises a third and a fourth label attachment regions, which are non-overlapping with the other label attachment regions, each comprising a DNA sequence hybridized to an RNA molecule that is attached to one or more detectable labels that emit light.
7. The method of claim 6 , wherein the first nucleic acid molecule further comprises at least one additional label attachment region, which is non-overlapping with the other label attachment regions, comprising a DNA sequence hybridized to an RNA molecule that is not attached to a detectable label.
8. The method of claim 6 , wherein the first nucleic acid molecule comprises at least three additional label attachment regions, which are non-overlapping with the other label attachment regions, each comprising a DNA sequence hybridized to an RNA molecule that is not attached to a detectable label.
9. The method of claim 8 , wherein an additional label attachment region comprising a DNA sequence hybridized to an RNA molecule that is not attached to a detectable label is located between a pair of label attachment regions in which each member of the pair comprises a DNA sequence hybridized to an RNA molecule that is attached to one or more detectable labels.
10. The method of claim 9 , wherein an additional label attachment region comprising a DNA sequence hybridized to an RNA molecule that is not attached to a detectable label is located between each pair of label attachment regions in which each member of the pair comprises a DNA sequence hybridized to an RNA molecule that is attached to one or more detectable labels.
11. The method of claim 1 , wherein the force is a direct current electrical field.
12. The method of claim 1 , wherein the first moiety or the second moiety selectively binds to the substrate via one or more non-covalent bonds.
13. The method of claim 1 , wherein the first moiety or the second moiety selectively binds to the substrate via one or more covalent bonds.
14. The method of claim 1 , wherein the force is sufficient to orient the polynucleotide complex.
15. The method of claim 14 , wherein the extended polynucleotide complex is immobilized in an oriented state.
16. The method of claim 1 , wherein the target nucleic acid is single stranded.
17. The method of claim 1 , wherein the target nucleic acid comprises two complementary strands.
18. The method of claim 1 , wherein the target nucleic acid is DNA or RNA.
19. The method of claim 1 , wherein the first and second RNA molecules each comprise four or more aminoallyl-modified UTP nucleotides.
20. The method of claim 6 , wherein the first and second RNA molecules and the RNA molecules hybridized to the third or the fourth label attachment regions each comprise four or more aminoallyl-modified UTP nucleotides.
21. The method of claim 19 , wherein one or more fluorophore labels is attached to each aminoallyl-modified UTP nucleotide.
22. The method of claim 20 , wherein one or more fluorophore labels is attached to each aminoallyl-modified UTP nucleotide.
23. The method of claim 1 , wherein the first nucleic acid molecule or the second nucleic acid molecule hybridizes to a terminus of the target nucleic acid.
24. The method of claim 23 , wherein the first nucleic acid molecule and the second nucleic acid molecules each hybridize to a terminus of the target nucleic acid.
25. The method of claim 1 , wherein the first nucleic acid molecule or the second nucleic acid molecule hybridizes to a non-terminus of the target nucleic acid.
26. The method of claim 25 , wherein the first nucleic acid molecule and the second nucleic acid molecules each hybridize to a non-terminus of the target nucleic acid.
27. The method of claim 1 , wherein the first moiety and the second moiety are each selected from the group consisting of ligands, antigens, carbohydrates, nucleic acids, receptors, lectins, and antibodies.
28. The method of claim 1 , wherein the first moiety or the second moiety are each selected from the group consisting of biotin, digoxigenin, FITC, avidin, streptavidin, antidigoxigenin, and anti-FITC.
29. The method of claim 1 , wherein the first moiety or the second moiety are each selected from the group consisting of succinamides, amines, aldehydes, epoxies, and thiols.
30. The method of claim 1 , wherein the substrate comprises a ligand, an antigen, a nucleic acid, a receptor, or an antibody.
31. The method of claim 30 , wherein the substrate comprises biotin, digoxigenin, FITC, avidin, streptavidin, antidigoxigenin, or anti-FITC.
32. The method of claim 1 , wherein the substrate comprises succinamides, amines, aldehydes, epoxies, or thiols.
33. The method of claim 1 , wherein the substrate is comprised of nitrocellulose, nylon, glass, a polymer, a gel, dextran, cellulose, or latex.
34. The method of claim 1 , wherein the substrate is selected from the group consisting of a membrane, a bead, a filter, a porous material, and a glass surface.
35. The method of claim 34 , wherein the membrane is comprised of nitrocellulose or nylon.
36. The method of claim 34 , wherein the filter is comprised of nitrocellulose or nylon.
37. The method of claim 34 , wherein the porous material is comprised of an acrylic, styrene methyl methacrylate copolymer, or ethylene/acrylic acid.
38. The method of claim 1 , wherein the substrate comprises a form selected from the group consisting of a disk, a slab, a strip, a bead, a submicron particle, a coated magnetic bead, a gel pad, a microtiter well, a slide, a membrane, and a frit.
39. The method of claim 1 , wherein the substrate is coated with streptavidin, biotinylated BSA, aldehyde, or epoxy.