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Details for Patent: 5,861,254

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Details for Patent: 5,861,254

Title: Flow cell SELEX
Abstract:Described herein are methods for improved partitioning between high and low affinity nucleic acid ligands identified through the SELEX method, termed Flow Cell SELEX. The Flow Cell SELEX method achieves partitioning between high and low affinity nucleic acid ligands using surface plasmon resonance technology. The method of the present invention presents a new and powerful approach to select nucleic acid ligands by providing a partitioning method which 1) enables a significant increase in the efficiency of partitioning from traditional partitioning methods used in SELEX, 2) allows for real time monitoring of the partitioning of the high affinity ligands from the low affinity ligands 3) allows for the ability to select for a nucleic acid ligand having specific kinetic properties, 4) does not rely on radiolabeling or other means of tagging for detection, and 5) allows for use of smaller amounts of target than in traditional methods of SELEX.
Inventor(s): Schneider; Daniel J. (Broomfield, CO), Vanderslice; Rebecca (Boulder, CO), Gold; Larry (Boulder, CO)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO)
Filing Date:Jan 31, 1997
Application Number:08/792,075
Claims:1. A method for identifying a nucleic acid ligand to a target from a candidate mixture of nucleic acids comprising

a) immobilizing a target on a sensor chip;

b) preparing a candidate mixture of nucleic acids;

c) passing the candidate mixture of nucleic acids over the surface of the sensor chip, wherein nucleic acids having increased affinity to the immobilized target relative to the candidate mixture bind to the immobilized target;

d) passing a wash buffer over the sensor chip at a determined flow rate to remove unbound nucleic acids;

e) passing an eluent over the sensor chip to elute the nucleic acids which are bound to the immobilized target; and

f) amplifying the eluted nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby a nucleic acid ligand to a target is identified.

2. The method of claim 1 further comprising g) repeating steps c), d), e) and f).

3. The method of claim 1 wherein the flow rate of the candidate mixture over the sensor chip is adjusted to optimize for the selection of nucleic acids having a fast on-rate and a slow off-rate.

4. The method of claim 3 wherein the flow rate of the wash buffer over the sensor chip is adjusted to optimize for the selection of nucleic acids having a fast on-rate and a slow off-rate.

5. The method of claim 1 wherein the composition of the wash buffer in step d) is adjusted to optimize for the selection of nucleic acids having a slow off-rate.

6. The method of claim 5 wherein the composition of said eluent comprises solubilized form of the immobilized target, wherein nucleic acids bound to the immobilized target having fast off-rates bind to the soluble target, wherein the selection for nucleic acids bound to the immobilized target having slow off-rates is enhanced.

7. The method of claim 1 wherein said candidate mixture of nucleic acids comprises single-stranded nucleic acids.

8. The method of claim 7 wherein said single stranded nucleic acids are ribonucleic acids.

9. The method of claim 7 wherein said single stranded nucleic acids are deoxyribonucleic acids.

10. The method of claim 8 wherein said candidate mixture of nucleic acids comprises 2'-modified ribonucleic acids.

11. The method of claim 8 wherein said 2'-modified ribonucleic acids comprise 2'-fluoro (2'-F) modified nucleic acids.

12. A method for identifying a nucleic acid ligand to a target from a candidate mixture of nucleic acids comprising:

a) immobilizing a target on a sensor chip;

b) preparing a candidate mixture of nucleic acids;

c) dividing said candidate mixture into a plurality of aliquots;

d) passing a first aliquot over the surface of the sensor chip, wherein nucleic acids having increased affinity to the immobilized target relative to the candidate mixture are bound to the immobilized target;

e) passing a wash buffer over the sensor chip to remove unbound nucleic acids;

f) repeating steps d) and e) until all of the aliquots have been injected;

g) passing an eluent over the sensor chip at a determined flow rate to elute the nucleic acids which are bound to the immobilized target; and

h) amplifying the eluted nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby a nucleic acid ligand to a target is identified.

13. A method for identifying a nucleic acid ligand to a target from a candidate mixture of nucleic acids comprising:

a) immobilizing a target on a sensor chip;

b) preparing a candidate mixture of nucleic acids;

c) dividing said candidate mixture into a plurality of aliquots;

d) passing a first aliquot over the surface of the sensor chip in a controlled flow, wherein nucleic acids having increased affinity to the immobilized target relative to the candidate mixture are bound to the immobilized target;

e) washing the sensor chip with a buffer to remove unbound nucleic acids;

f) eluting the nucleic acids which are bound to the immobilized target;

g) repeating steps d), e), and f) until all of the aliquots of the candidate mixture have been injected;

h) combining the eluted nucleic acids from step f); and

i) amplifying the combined eluted nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby a nucleic acid ligand to a target molecule is identified.
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