Methods and compositions for processes of rapid selection and production of nucleic acid aptamers

• US 9,273,345 B2
• Filed: 12/27/2007
• Issued: 03/01/2016
• 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.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US11/965,039
• Publication Number:
  US 20090004644A1
• Time in Patent Office: 2,987 Days
• Field of Search: None
• US Class Current: 1/1
• 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 comprising at least one double-stranded nucleic acid molecule, the at least one double-stranded nucleic acid molecule comprising:

a reporter moiety bound to a fully-annealed first strand of the double- stranded nucleic acid molecule wherein the reporter moiety is capable of generating a signal; and

a signal reducing moiety bound to a second, separate and fully complementary strand of the first nucleic acid strand wherein the signal reducing moiety is capable of reducing the signal produced by the reporter moiety,

wherein the first and the second strand of the double-stranded nucleic acid molecule are the same length, and wherein either the first nucleic acid strand or the second nucleic acid strand of the double-stranded nucleic acid molecule is an aptamer and binds in a non-complementary manner to a target agent.

2. The composition of claim 1, further comprising a solid substrate, wherein the solid substrate is attached to either the first strand or the second strand of the double-stranded nucleic acid molecule.

3. The composition of claim 2, wherein the solid substrate is bound to the first strand.

4. The composition of claim 2, wherein the solid substrate comprises beads, microbeads, particles, microparticles, nanoparticles or a combination of two or more thereof.

5. The composition of claim 2, 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, microspheres with surface primary amine groups, 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.

6. 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.

7. The composition of claim 1, wherein the double-stranded nucleic acid molecule ranges from 10 to 200 base pairs in length.

8. The composition of claim 1, wherein the target agent is chosen from a virus, a yeast, a spore, a bacterium, a biomarker for disease, a biomarker for disease progression or a combination thereof.

9. The composition of claim 1, wherein the target agent comprises a non-nucleic acid target agent and the non-nucleic acid target is selected from the group consisting of, a whole organism comprising bacteria, yeast or virus;

protein, peptide, carbohydrate, polysaccharide, glycoprotein, lipid, hormone, receptor, antigen, allergen, antibody, substrate, metabolite, cofactor, enzyme, metal ion, inhibitor, drug, pharmaceutical, nutrient, toxin, poison, explosive, pesticide, chemical warfare agent, biohazardous agent, prion, radioisotope, vitamin, heterocyclic aromatic compound, carcinogen, mutagen, narcotic, amphetamine, barbiturate, hallucinogen, waste product, or a contaminant.

10. The composition of claim 1, wherein the target agent is a protein.

11. The composition of claim 1, wherein at least one strand of the double-stranded nucleic acid molecule interacts with the target agent by secondary structure features.

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

15. A method for producing the at least one double-stranded nucleic acid molecule of claim 1, the method comprising:

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

b) contacting a second reagent moiety with a second nucleic acid molecule mixture to form a second reagent moiety-nucleic acid molecule complex comprising nucleic acid molecules 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;

wherein either of the first nucleic acid molecule mixture or the second nucleic acid molecule mixture comprises at least one aptamer, and wherein the other of the first nucleic acid molecule mixture or second nucleic acid molecule mixture comprises a nucleic acid molecule fully complementary to and equal in length to the at least one aptamer; and

c) combining the first nucleic acid molecule mixture and the second nucleic acid molecule mixture under conditions such that the at least one aptamer fully anneals to the complementary nucleic acid molecule.

16. The method of claim 15, wherein the first reagent moiety, second reagent moiety or combination thereof are in solution.

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

13. A composition comprising, a quenching agent moiety bound to at least one separate and fully complementary strand of a fully-annealed dsDNA molecule, wherein absorption wavelength of the quenching agent moiety overlaps with emission wavelength of an agent capable of generating a signal, wherein one of the separate and fully complementary strands of the fully-annealed dsDNAmolecule is an aptamer and binds in a non-complementary manner to a non-nucleic acid target agent, and wherein the strands of the dsDNA molecule are the same length.

14. The composition of claim 13, wherein the agent capable of generating a signal is a quantum dot.