Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 99, Issue 18, Pages 11616-11621Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.182095699
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Funding
- NIGMS NIH HHS [F32 GM017438, GM53036, GM17438] Funding Source: Medline
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Aptamers, RNA sequences that bind to target ligands, are typically isolated by in vitro selection from RNA libraries containing completely random sequences. To see whether higher-affinity aptamers can be isolated from partially structured RNA libraries, we selected for aptamers that bind GTP, starting from a mixture of fully random and partially structured libraries. Because stem-loops are common motifs in previously characterized aptamers, we designed the partially structured library to contain a centrally located stable stem-loop. We used an off-rate selection protocol designed to maximize the enrichment of high-affinity aptamers. The selection produced a surprisingly large number of distinct sequence motifs and secondary structures, including seven different aptamers with K(d)s ranging from 500 to 25 nanomolar. The engineered stem-loop was present in the three highest affinity aptamers, and in 12 of 13 independent isolates with a single consensus sequence, suggesting that its inclusion increased the abundance of high-affinity aptamers in the starting pool.
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