Method and apparatus for the automated generation of nucleic acid ligands

• US 20020064780A1
• Filed: 03/22/2001
• Published: 05/30/2002
• Est. Priority Date: 06/11/1990
• Status: Active Grant

• Current Assignee: Somalogic Incorporated
• Original Assignee: Somalogic Incorporated
• Inventors: Gold, Larry, Jenison, Robert D., Schneider, Daniel J., Zichi, Dominic A.
• Granted Patent: US 6,716,580 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1048 SELEX

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 robotic manipulator controlled by a computer. The invention also includes methods and reagents to obviate the need for size-fractionation of amplified candidate nucleic acids before beginning the next round of the SELEX process.

Claims

1. A method for the identification of a nucleic acid ligand from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target, each said nucleic acid in said candidate mixture comprising fixed sequence regions, the method comprising:

a) contacting the candidate mixture with the target 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; and

c) amplifying the increased affinity nucleic acids using the polymerase chain reaction (PCR) with primers complementary to said fixed sequence regions to yield a ligand enriched mixture of nucleic acids, wherein the 5′

ends of said primers are attached to tail sequences having a lower melting temperature (Tm) than said primers, wherein the polymerase chain reaction comprises a denaturation step, a primer annealing step, and a primer extension step, and wherein said primer annealing step and said primer extension step are performed at a temperature higher than the melting temperature of said tail sequences;

d) repeating steps a) through c) using the ligand enriched mixture of each successive repeat as many times as required to yield a desired level of increased ligand enrichment;

wherein a nucleic acid ligand is identified.

2. The method of claim 1 wherein steps (a)-(c) are performed at one or more work stations on a work surface by a robotic manipulator controlled by a computer.

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

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

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

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

7. The method of claim 3 wherein said solid support is a paramagnetic bead and wherein the partitioning of said paramagnetic bead is performed by a magnetic bead separator controlled by said computer.

8. The method of claim 1 wherein each nucleic acid in said candidate mixture of nucleic acid ligands has a tail sequence at both the 5′

terminus and the 3′

terminus, wherein each tail sequence has a lower melting temperature (Tm) than said fixed sequence regions.

9. The method of claim 8 wherein said candidate nucleic acid mixture comprises a nucleic acid selected from the group consisting of SEQ. ID. NO:

3, SEQ. ID. NO;

6, SEQ. ID. NO;

7, SEQ. ID. NO;

8, SEQ. ID. NO;

9, SEQ. ID. NO;

10, SEQ. ID. NO;

11, SEQ. ID. NO;

12, SEQ. I). NO;

13, and SEQ. ID. NO;

14.

10. The method of claim 9 wherein said candidate nucleic acid mixture is SEQ. ID. NO:

7, and wherein said primers are SEQ. ID. NO. 16 and SEQ. ID. NO;

25.

11. The method of claim 2 wherein said computer makes a measurement of the amount of amplified product and calculates a value for the initial concentration of nucleic acid ligand eluted from target using said measurement.

12. The method of claim 11 wherein said computer adjusts the reaction conditions of steps (a)-(c) in a predetermined manner in response to said value.

13. The method of claim 11 wherein step c) is performed in the presence of a fluorescent nucleic acid-intercalating dye, said dye undergoing a change in fluorescence intensity upon binding double-stranded DNA, and wherein said measurement of the amount of amplified product is made by quantitating the fluorescence signal from said dye.

14. An automated machine carrying out the method of claim 1.

16. The method of claim 15 wherein steps (a)-(e) are performed at one or more work stations on a work surface by a robotic manipulator controlled by a computer.

18. The method of claim 17 wherein steps a)-g) are carried out by automated machines controlled by a computer.

20. The method of claim 19 further comprising the step:

e) repeating steps a) through d) using the ligand enriched mixture of each successive repeat as many times as required to yield a desired level of increased ligand enrichment.

21. The method of claim 19 wherein step a) comprises:

(i) contacting said candidate mixture of nucleic acids with a nucleic acid primer comprising sequence from said 3′

terminal fixed sequence region, wherein said primer is attached to the surface of a solid support;

(ii) discarding nucleic acids in said candidate mixture that anneal to said primer on the surface of said solid support.

15. A method for identifying nucleic acid ligands of a target molecule from a candidate mixture of nucleic acids, the method comprising the steps:

a) preparing a candidate mixture of nucleic acids, said nucleic acids comprising photoreactive groups and fixed sequence regions;

b) contacting said candidate mixture with said target molecule, wherein nucleic acid sequences having increased affinity to the target molecule relative to the candidate mixture form nucleic acid-target molecule complexes;

c) irradiating said candidate mixture, wherein said nucleic acid-target molecule complexes photocrosslink;

d) partitioning the crosslinked nucleic acid-target molecule complexes from free nucleic acids in the candidate mixture; and

e) amplifying the nucleic acids that crosslinked to the target molecule to yield a mixture of nucleic acids enriched in sequences that are capable of photocrosslinking the target molecule, wherein the amplification is performed using the polymerase chain reaction (PCR) with primers complementary to said fixed sequence regions to yield amplified increased affinity nucleic acids, wherein the 5′

ends of said primers are attached to tail sequences having a lower melting temperature (Tm) than said primers, wherein the polymerase chain reaction comprises a denaturation step, a primer annealing step, and a primer extension step, and wherein said primer annealing step and said primer extension step are performed at a temperature higher than the melting temperature of said tail sequences;

f) repeating steps a) through e) using the ligand enriched mixture of each successive repeat as many times as required to yield a desired level of increased ligand enrichment;

wherein a nucleic acid ligand is identified

17. A method for identifying a photocrosslinking nucleic acid ligand of a protein from a candidate mixture of nucleic acids comprising fixed sequence regions, said method comprising:

a) contacting said candidate mixture with said protein, wherein nucleic acids having increased affinity to the protein relative to the candidate mixture form nucleic acid-protein complexes with the protein;

b) partitioning the complexed increased affinity nucleic acids from the remainder of the candidate mixture;

c) amplifying the increased affinity nucleic acids using the polymerase chain reaction (PCR) with primers complementary to said fixed sequence regions to yield amplified increased affinity nucleic acids, wherein the 5′

ends of said primers are attached to tail sequences having a lower melting temperature (Tm) than said primers, wherein the polymerase chain reaction comprises a denaturation step, a primer annealing step, and a primer extension step, and wherein said primer annealing step and said primer extension step are performed at a temperature higher than the melting temperature of said tail sequences;

d) incorporating photoreactive groups into said amplified increased affinity nucleic acids;

e) repeating step a;

f) irradiating said increased affinity nucleic acids, wherein said nucleic acid- protein complexes photocrosslink;

g) repeating steps c) and d); and

h) identifying a photocrosslinking nucleic acid ligand to the protein.

19. A method for the identification of a nucleic acid ligand from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target, each said nucleic acid in said candidate mixture comprising a 3′

terminal fixed sequence region, a 5′

terminal fixed sequence region, and one or more random sequence regions, the method comprising;

a) depleting said candidate mixture of nucleic acids in which at least one of said random sequence regions is complementary in sequence to said 3′

terminal fixed sequence region;

b) contacting the candidate mixture with the target 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;

c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and

d) amplifying the increased affinity nucleic acids;

wherein a nucleic acid ligand is identified.

22. A method of amplifying a mixture of nucleic acids, each nucleic acid in said mixture comprising a 3′

terminal fixed sequence region, a 5′

terminal fixed sequence region, and at least one random sequence region, the method comprising;

a) providing primers complementary to said fixed sequence regions, wherein the 5′

end of each primer is attached to a tail sequence having a lower melting temperature (Tm) than said primers;

b) performing the Polymerase Chain Reaction (PCR) using said primers attached to said tail sequences, wherein the primer annealing step and the primer extension step of the Polymerase Chain Reaction are performed at a temperature higher than the melting temperature of the tail sequences, whereby the formation of self-priming intramolecular and intermolecular duplexes by said nucleic acids in said mixture is prevented.

25. The method of claim 24 wherein each nucleic acid in (a) has a tail sequence at both the 5′

terminus and the 3′

terminus, wherein each tail sequence has a lower melting temperature (Tm) than said fixed sequence regions.

23. A method of removing from a mixture of nucleic acids comprising a 3′

terminal fixed sequence region, a 5′

terminal fixed sequence region and at least one random sequence region those nucleic acids capable of forming intramolecular duplexes between the 3′

region and the random region of the same nucleic acid, or intermolecular duplexes between the 3′

region of one nucleic acid and the random region of another, the method comprising;

(a) contacting said mixture of nucleic acids with a nucleic acid primer comprising sequence from said 3′

terminal fixed sequence region, wherein said primer is attached to the surface of a solid support;

(b) discarding nucleic acids in said mixture that anneal to said primer on the surface of said solid support.

24. A method for preparing a nucleic acid having a specific function, the method comprising:

(a) preparing a test mixture of nucleic acids, each nucleic acid in said test mixture comprising fixed sequence regions and at least one random sequence region;

(b) selecting those nucleic acids in said test mixture having said specific function;

(c) partitioning those nucleic acids selected in (b) from the remainder of the test mixture;

(d) amplifying the partitioned nucleic acids in (c) using the polymerase chain reaction (PCR) with primers complementary to said fixed sequence regions to yield an amplified test mixture enriched for those nucleic acids having said specific function, wherein the 5′

ends of said primers are attached to tail sequences having a lower melting temperature (Tm) than said primers, wherein the polymerase chain reaction comprises a denaturation step, a primer annealing step, and a primer extension step, and wherein said primer annealing step and said primer extension step are performed at a temperature higher than the melting temperature of said tail sequences;

(e) repeating steps a) through d) using the amplified test mixture of each successive repeat as many times as required to yield a desired level of increased enrichment for nucleic acids having said specific function, wherein a nucleic acid having said specific function is identified.