Method for the automated generation of nucleic acid ligands

• US 6,569,620 B1
• Filed: 01/19/1999
• Issued: 05/27/2003
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Somalogic Incorporated
• Original Assignee: Somalogic Incorporated
• Inventors: Schneider, Daniel J., Gold, Larry, Jenison, Robert D., Zichi, Dominic A.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): FORMAN, BETTY J
• Application Number:
  US09/232,946
• Time in Patent Office: 1,589 Days
• Field of Search: 435/6, 435/91.2, 435/287.2, 435/288.3, 435/288.4, 435/288.7, 422/63, 422/68.1, 536/23.1
• US Class Current: 435/6.11
• 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: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2330/31 Libraries, arrays
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2541/101 Selex
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads

Abstract

The present invention includes a method and device for performing automated SELEX. The steps of the SELEX process are performed at one or more work stations on a work surface by a cartesian robotic manipulator controlled by a computer.

Claims

1. A method for the automated identification of a nucleic acid ligand from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target comprising:

a) adding the candidate mixture and the target in a predetermined ratio to a reaction vessel at a work station on a work surface using a cartesian robotic manipulator, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture using said robotic manipulator;

c) amplifying the increased affinity nucleic acids to yield an amplified, ligand-enriched mixture of nucleic acids by;

i) adding primers and polymerase chain reaction reagents to the reaction vessel using said robotic manipulator;

ii) thermally-cycling the reaction vessel using a thermal cycler while simultaneously measuring the amount of amplified product in said reaction vessel using a measuring device; and

iii) calculating the amount of increased affinity nucleic acids partitioned at step b) using the measurement of the amount of amplified product obtained from said measuring device;

d) adjusting the reaction conditions of steps a)-c) in a predetermined manner in response to the amount of nucleic acid ligand calculated at step c) iii); and

e) repeating steps a)-d) at least once, wherein the adjustments performed at step d) control the stringency of each successive repeat;

wherein said robotic manipulator, said thermocycler, and said measuring device are automatically controlled by a computer during steps a)-e), and wherein said computer automatically calculates the amount of increased affinity product at step c) and automatically adjusts the reaction conditions at step d);

whereby a nucleic acid ligand of said target is identified automatically.

2. The method of claim 1 wherein said target is attached to a solid support, and wherein step (b) is accomplished by automatically partitioning said solid support from said candidate mixture using said robotic manipulator.

3. The method of claim 2 wherein said solid support is a multi-well microtitre plate.

4. The method of claim 3 wherein said plate is comprised of polystyrene.

5. The method of claim 4 wherein said target is attached to said plate by hydrophobic interactions.

6. The method of claim 2 wherein said solid support is a paramagnetic bead and wherein the partitioning of said paramagnetic bead is performed automatically by a magnetic bead separator controlled by said computer.

7. The method of claim 1 wherein said primers are labeled with fluorophores and quenching groups at nucleotide positions that move relative to one another when said primers become incorporated into amplified product, such that the fluorescence emission profiles of said primers change upon incorporation into amplified product, and wherein said measuring device makes the measurement of the amount of amplified product by detecting said change.

8. The method of claim 7 wherein at least one of said primers comprises:

(a) a single stranded DNA molecule complementary to one of said fixed sequence regions;

(b) a stem-loop structure attached to the 5′

end of said single stranded DNA molecule, said stem comprising a fluorophore and a quenching agent located at nucleotide positions on opposite sides of the stem of said stem-loop structure, said nucleotide positions located sufficiently close to one another such that the fluorescent signal from said fluorophore is substantially quenched by said quenching agent;

wherein the extension of the 3′

end of candidate nucleic acid ligands that anneal to said primer during said Polymerase Chain Reaction disrupts said stem structure, wherein said fluorescent group is no longer quenched by said quenching group.

9. The method of claim 8 wherein said primer is selected from the group consisting of:

10. The method of claim 1, wherein the candidate mixture of nucleic acids is ribonucleic acids, and the method comprises:

a) adding the candidate mixture and the target in a predetermined ratio to a reaction vessel at a work station on a work surface using a cartesian robotic manipulator, wherein ribonucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity ribonucleic acids from the remainder of the candidate mixture using said robotic manipulator;

c) reverse transcribing the increased affinity ribonucleic acids to produce DNA template by adding reverse transcription reagents to said reaction vessel using said robotic manipulator;

d) amplifying the DNA template by;

i) adding primers and polymerase chain reaction reagents to the reaction vessel using said robotic manipulator;

ii) thermally-cycling said reaction vessel using a thermal cycler while simultaneously measuring the amount of amplified product using a measuring device;

iii) calculating the amount of increased affinity ribonucleic acids partitioned at step b) using the measurement of the amount of amplified product obtained from the measuring device;

e) transcribing the amplified DNA to RNA by adding transcription reagents to the reaction vessel using said robotic manipulator;

f) purifying the transcribed RNA from the amplified DNA by adding RNA specific primers bound to a solid support using said robotic manipulator, whereby only the transcribed RNA binds to the primers;

g) partitioning the transcribed RNA from the amplified DNA using said robotic manipulator;

h) adjusting the reaction conditions of steps a)-g) in a predetermined manner in response to the amount of nucleic acid ligand calculated at step d) iii); and

i) repeating steps a)-h) at least once using the partitioned transcribed RNA of step g) as candidate mixture in step a), wherein the adjustments performed at step h) control the stringency of each successive repeat;

wherein said robotic manipulator, said thermocycler, and said measuring device are automatically controlled by a computer during steps a)-i), and wherein said computer automatically calculates the amount of increased affinity product at step d) and automatically adjusts the reaction conditions at step h);

whereby a ribonucleic acid ligand of said target is automatically identified.