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Last Updated: March 28, 2024

Details for Patent: 5,773,598


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Title: Systematic evolution of ligands by exponential enrichment: chimeric selex
Abstract:Methods are disclosed for producing chimeric nucleic acid molecules with two or more functionalities. A chimeric library is generated in which individual chimeric molecules combine the functions or characteristics of two or more parent libraries, each parent library having been selected through the SELEX procedure for a specific function or feature. The chimeric molecules of this invention are useful in a variety of ways, including having improved affinities for a target molecule, enhancing assembly of multi-component molecules, and promoting reactions between two molecules.
Inventor(s): Burke; Donald (Boulder, CO), Tarasow; Ted (Boulder, CO), Eaton; Bruce E. (Boulder, CO), Gold; Larry (Boulder, CO)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO)
Filing Date:Jun 05, 1995
Application Number:08/464,102
Claims:1. A chimeric nucleic acid ligand having high binding affinity for first and second target molecules identified according to a method comprising:

a) preparing a chimeric nucleic acid ligand library generated according to a method comprising:

i) generating a first library of nucleic acid ligands selected through the SELEX procedure for binding to said first target molecule, said nucleic acid ligands having a 3' fixed sequence, and producing the double-stranded form of said first library of nucleic acid ligands;

ii) generating a second library of nucleic acid ligands selected through the SELEX procedure for binding to said second target molecule, said nucleic acid ligands having a 5' fixed sequence identical to the 3' fixed sequence of the nucleic acid ligands of said first library, and producing the double-stranded form of said second library of nucleic acid ligands;

iii) mixing said first and second libraries under conditions which promote interlibrary annealing;

iv) forming chimeric nucleic acid ligands by enzymatically extending the recessed 3' ends while copying the 5' extensions of each annealed pair to yield a double-stranded nucleic acid ligand library; and

v) amplifying said double-stranded chimeric nucleic acid ligand library;

b) contacting said amplified chimeric nucleic acid ligand library with said first and second target molecules, wherein chimeric nucleic acid ligands having an increased affinity to said first and second target molecules may be partitioned from the remainder of the chimeric nucleic acid ligand library;

c) partitioning said increased affinity chimeric nucleic acid ligands from the remainder of said chimeric nucleic acid ligand library; and

d) amplifying said increased affinity chimeric nucleic acid ligands to yield a mixture of chimeric nucleic acid ligands enriched in members which bind to said first and second target molecules, whereby a chimeric nucleic acid ligand having high binding affinity to said first and second target molecules is identified.

2. A chimeric nucleic acid ligand having high binding affinity for first and second target molecules identified according to a method comprising:

a) preparing a chimeric nucleic acid ligand library generated according to a method comprising:

i) generating a first library of nucleic acid ligands selected through the SELEX procedure which bind to said first target molecule, said nucleic acid ligands having a 3' fixed sequence and a 5'-monophosphate;

ii) generating a second library of nucleic acid ligands selected through the SELEX procedure which bind to said second target molecule;

iii) mixing said first and second libraries under conditions where said nucleic acid ligands of said first library are enzymatically or chemically joined to said nucleic acid ligands of said second library to form chimeric nucleic acid ligands; and

iv) amplifying said chimeric nucleic acid ligands, to yield said chimeric nucleic acid ligand library;

b) contacting said amplified chimeric nucleic acid ligand library with said first and second target molecules, wherein chimeric nucleic acid ligands having an increased affinity to said first and second target molecules may be partitioned from the remainder of the chimeric nucleic acid ligand library;

c) partitioning said increased affinity chimeric nucleic acid ligands from the remainder of said chimeric nucleic acid ligand library; and

d) amplifying said increased affinity chimeric nucleic acid ligands to yield a mixture of chimeric nucleic acid ligands enriched in members which bind to said first and second target molecules, whereby a chimeric nucleic acid ligand having high binding affinity to said first and second target molecules is identified.

3. A chimeric nucleic acid ligand comprising 3'--3' linked nucleic acid ligands, said chimeric nucleic acid having high binding affinity for a target molecule identified according to a method comprising:

a) preparing a chimeric nucleic acid library generated according to a method comprising:

i) generating a first library of nucleic acid ligands selected through the SELEX procedure for binding to said target molecule wherein each of said nucleic acid ligands is modified at the 3' end to facilitate linking of members of said first library with each other; and

ii) chemically reacting members of said first library of nucleic acid ligands to form chimeric 3'--3'-linked nucleic acid ligands which comprise said chimeric nucleic acid library;

b) contacting said chimeric nucleic acid ligand library with said target molecule, wherein chimeric 3'--3' linked nucleic acid ligands having an increased affinity to said target molecule may be partitioned from the remainder of the chimeric nucleic acid ligand library

c) partitioning said increased affinity chimeric 3'--3' linked nucleic acid ligands from the remainder of said chimeric nucleic acid library;

d) amplifying said increased affinity chimeric 3'--3' linked nucleic acid ligands to yield a mixture of nucleic acid ligands representing each half of said chimeric 3'--3' linked nucleic acid ligands, enriched in nucleic acid ligands having increased affinity to said target molecule; and

e) chemically reacting said mixture of amplified increased affinity nucleic acid ligands to form increased affinity chimeric 3'--3'-linked nucleic acid ligands, whereby a chimeric nucleic acid ligand comprising 3'--3' linked nucleic acid ligands having high binding affinity to said target molecule is identified.

4. The chimeric nucleic acid molecule of claim 1 identified according to a method further comprising between step a)v) and step b), the step:

vi) transcription of said double-stranded nucleic acid ligand library to yield a single-stranded RNA chimeric nucleic acid ligand library.

5. The chimeric nucleic acid molecule of claim 1 identified according to a method further comprising between step a)v) and step b), the step:

vi) separating the strands of said double-stranded nucleic acid ligands to yield a single-stranded nucleic acid chimeric nucleic acid ligand library.

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