| 摘要 |
A method for identifying nucleic acid ligands to target molecules using the SELEX procedure. Nucleic acid candidate sequences contain photoreactive groups. After exposure of the nucleic acid sequences to the target molecule, nucleic acid-target molecule complexes are formed between nucleic acids having increased affinity to the target molecule and the target molecule. The complexes are irradiated such that photocrosslinks form between the photoreactive groups of the bound nucleic acids and the target molecule. The photocrosslinked complexes are separated from unbound nucleic acids, and the nucleic acids amplified to yield a ligand-enriched mixture of nucleic acids. Described herein are methods for improved partitioning between high and low affinity nucleic acid ligands identified through the SELEX method, termed solution SELEX. The solution SELEX method achieves partitioning between high and low affinity nucleic acid-target complexes through a number of methods, including (1) primer extension inhibition which results in differentiable cDNA products. Primer extension inhibition is achieved with the use of nucleic acid polymerases, including DNA or RNA polymerases, reverse transcriptase, and Qbeta-replicase; (2) exonuclease hydrolysis inhibition which results in only the highest affinity ligands amplifying during PCR. This is achieved with the use of any 3'->5' double-stranded exonuclease; (3) linear to circle formation to generate molecules amplifiable during PCR; or (4) PCR amplification of single-stranded nucleic acids. A central theme of the method of the present invention is that the nucleic acid candidate mixture is screened in solution and results in preferential amplification of the highest affinity RNA ligand or catalytic RNA.
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