METHODS AND COMPOSITIONS FOR PROCESSES OF RAPID SELECTION AND PRODUCTION OF NUCLEIC ACID APTAMERS

• US 20090004644A1
• Filed: 12/27/2007
• Published: 01/01/2009
• Est. Priority Date: 12/28/2006
• Status: Active Grant

• Current Assignee: Conceptual MindWorks Incorporated
• Original Assignee: Conceptual MindWorks Incorporated
• Inventors: Kiel, Johnathan L., Holwitt, Eric A., Fan, Michael (Maomian), Roper, Shelly D.
• Granted Patent: US 9,273,345 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Embodiments herein relate to compositions and methods for making and using aptamers, for example, DNA aptamers (DCEs) and/or RNA aptamers. In some embodiments, methods relate to making and amplifying target DCEs. In certain embodiments, methods for making capture elements or aptamers concern using a reporter moiety and signal reducing moiety prior to amplifying a target-specific capture element. In some embodiments, methods disclosed herein may be used to rapidly generate large quantities of aptamers such as DCEs directed to a particular target agent. Some embodiments relate to systems for performing automated generation of aptamers.

Claims

1. A composition of nucleic acid sequences comprising:

a reporter moiety bound to a first strand of the nucleic acid sequence wherein the reporter moiety is capable of generating a signal; and

a signal reducing moiety bound to a second complimentary strand of the first nucleic acid sequence strand wherein the signal reducing moiety is capable of reducing the signal produced by the reporter moiety.

2. The composition of claim 1, wherein the reporter moiety is a quantum dot.

3. The composition of claim 1, further comprising a solid substrate, wherein the solid substrate is attached to at least one of the nucleic acid strands.

4. The composition of claim 3, wherein the solid substrate is bound to the first strand.

5. The composition of claim 3, wherein the solid substrate comprises beads, microbeads, particles, microparticles, nanoparticles or a combination of two or more thereof.

6. The composition of claim 3, wherein the solid substrate is chosen from carboxy-terminated beads, hydrazine terminated beads, Silica beads, IDA (iminodiacetic acid) modified beads, aldehyde modified beads, Epoxy activated beads, DADPA-modified beads, biodegradable polymeric beads, amino polybeads, amino-polystyrene particles, carboxyl-polystyrene particles, Epoxy-polystyrene particles, streptavidin coated beads, dimethylamino-polystyrene particles, hydroxy-polystyrene particles, colored particles, flow cytometry particles, sulfonate-polystyrene particles and a combination of two or more thereof.

7. The composition of claim 1, wherein the reporter moiety and the signal reducing moiety are a distance of 200 base pairs or less from one another.

8. The composition of claim 1, wherein the nucleic acid sequences are about 10 to about 200 base pairs in length.

9. The composition of claim 1, wherein the reporter moiety and the signal reducing moiety are covalently bound to the dsDNA.

10. The composition of claim 1, wherein the nucleic acid sequences are capable of forming a single-stranded nucleic acid-target agent complex when exposed to a target agent.

11. The composition of claim 1, wherein the target agent is chosen from a virus, a yeast, a spore, a bacterium, a disorder-associated molecule, a disease-progression molecule or a combination thereof.

12. The composition of claim 11, wherein the target agent comprises a nucleic acid sequence of the virus, the bacteria, the disorder-associated molecule, the disease-progression molecule or combination thereof.

13. A method for producing nucleic acid sequences for isolating DNA aptamers (DCEs) directed to bind a target agent comprising:

a) contacting a first reagent moiety with a first nucleic acid sequence mixture to form a first reagent moiety-nucleic acid sequence complex comprising nucleic acid sequences attached to first reagent moieties; and

b) contacting a second reagent moiety with a second nucleic acid sequence mixture to form a second reagent moiety-nucleic acid sequence complex comprising nucleic acid sequences attached to second reagent moieties;

wherein the first reagent moiety comprises a reporter moiety that is capable of generating a signal or a signal reducing moiety that is capable of reducing the signal from the reporter moiety and the second reagent moiety comprises the other of a reporter moiety or a signal reducing moiety.

14. The method of claim 13, wherein the first moiety, second moiety or combination thereof are in solution.

15. The method of claim 13, further comprising attaching a solid substrate to the strand bound to the reporter moiety.

16. The method of claim 13, contacting the first reagent moiety-nucleic acid sequence complex with a complimentary second nucleic acid sequence mixture for a time sufficient to allow double-stranded (ds) sequences to form then adding the second reagent moiety to the ds-sequences.

17. A method for producing nucleic acid aptamers comprising:

a) contacting a first reagent moiety with a first nucleic acid sequence mixture to form a first moiety-nucleic acid sequence complex comprising a nucleic acid that is attached to the first reagent moiety; and

b) contacting a second reagent moiety with a second nucleic acid sequence mixture to form a second moiety-nucleic acid sequence complex comprising a nucleic acid that is attached to the second reagent moiety wherein at least some of the nucleic acid sequences are complimentary to first nucleic acid sequences,

wherein the first reagent moiety comprises a qdot moiety that is capable of generating a signal or a quenching agent moiety that is capable of absorbing the signal from the qdot and the second reagent moiety comprises the other of a qdot moiety or a quenching agent moiety.

c) mixing the first moiety-nucleic acid sequence complex with the second moiety-nucleic acid sequence complex and allowing the formation of dsDNA (double-stranded DNA) complexes wherein the signal of the qdot moiety on one strand of the dsDNA is reduced when a quenching moiety is attached to a complimentary second strand on the dsDNA;

d) introducing a target agent to the dsDNA complexes, wherein the target agent binds to at least the first strand or the second strand of the dsDNA complex to form a target agent-single-stranded DCE (DNA capture element) complex (target agent-ssDCE complex); and

e) separating at least a portion of the target agent-ssDCE complex from the remainder of the complexes.

18. The method of claim 17, wherein the step of separating at least a portion of the target agent-ssDCE complex from the remainder of the complexes comprises associating the target agent-ssDCE complex with a solid substrate.

19. The method of claim 18, wherein the solid substrate is chosen from paramagnetic beads, magnetic beads, super paramagnetic beads, Reverse Phase magnetic beads, streptavidin coated beads, carboxy terminated beads, hydrazine terminated beads, Silica beads, IDA (iminodiacetic acid) modified beads, aldehyde modified beads, Epoxy activated beads, DADPA-modified beads, biodegradable polymeric beads, amino polybeads, amino-polystyrene particles, carboxyl-polystyrene particles, Epoxy-polystyrene particles, dimethylamino-polystyrene particles, hydroxy-polystyrene particles, colored particles, flow cytometry particles, sulfonate-polystyrene particles, or a combination thereof.

20. The method of claim 18, wherein associating the target agent-ssDCE complex with a solid substrate comprises binding the ssDCE to a complimentary strand of single-stranded DNA bound to the solid substrate.

21. The method of claim 17, further comprising f) amplifying the ssDCEs of the target agent-ssDCE complex.

22. The method of claim 21, further comprising annealing the amplified single-stranded sequences with a complimentary sequence to form double-stranded DCEs and optionally attaching the dsDCEs to a solid surface.

23. The method of claim 21, further comprising attaching the amplified ssDCE to a solid surface.

24. The method of claim 23, wherein the solid surface is chosen from glass, plastic, silicon-coated substrate, macromolecule-coated substrate, particles, beads, microparticles, microbeads, dipstick, magnetic beads, paramagnetic beads and a combination thereof.

25. A system for producing DNA aptamers comprising:

a) an element for inputting a first moiety reagent with a first mixture of nucleic acid sequences to form a first moiety reagent-nucleic acid sequence complex in a reaction vessel; and

b) an element for inputting a second moiety reagent with a second mixture of nucleic acid sequences to form a second moiety reagent-nucleic acid sequence complex in another reaction vessel;

wherein the first moiety reagent comprises a reporter moiety or a signal reducing moiety and the second reagent comprises the other of a reporter moiety or a signal reducing moiety.

26. The system of claim 25, further comprising c) an element for inputting contents of a) with contents of b);

or contents of b) with contents of a) and allowing the first and second nucleic acid sequences mixtures to anneal to form dsDNA complexes wherein the dsDNA reporter moiety signal is reduced in the presence of the signal reducing moiety.

27. The system of claim 26, further comprising an element for partitioning the dsDNA complexes from the remainder of the mixture.

28. The system of claim 26, further comprising an element for partitioning single-stranded nucleic acid sequences from the remainder of the mixture.

29. The system of claim 26, further comprising:

d) an element for inputting a target agent to the dsDNA complex to form a target agent-single-stranded nucleic acid complex wherein the target agent-single-stranded nucleic acid complexes are formed when the target agent binds to at least one strand of the dsDNA complex.

30. The system of claim 29, further comprising

e) an element for separating the target agent-single-stranded nucleic acid complex from the remainder of the complexes using a solid substrate wherein the target agent-single-stranded nucleic acid complex is bound directly or indirectly to the solid substrate;

and

f) an element for amplifying the single-stranded nucleic acid of the target agent-single-stranded nucleic acid complex bound to the solid substrate.

31. A composition comprising, a quenching agent moiety bound to a dsDNA sequence wherein adsorption wavelength of the quenching agent moiety overlaps with emission wavelength of an agent capable of generating a signal.

32. A kit for making aptamers comprising:

a reporter moiety composition; and

a signal reducing moiety composition.

33. The kit of claim 32, further comprising a mixture of nucleic acid sequences.

34. The kit of claim 32, wherein the reporter moiety composition comprises a quantum dot composition.

35. The kit of claim 32, wherein the signal reducing moiety composition comprises a quenching agent composition.

36. The kit of claim 32, further comprising a solid substrate for partitioning aptamers.

37. The kit of claim 36, wherein the solid substrate comprises magnetic beads.