Work

Isolation of high-affinity GTP aptamers from partially structured RNA libraries

Year: 2002

Type: article

Abstract: 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.

Source: Proceedings of the National Academy of Sciences

DOI: 10.1073/pnas.182095699

Authors: Jonathan H. Davis, Jack W. Szostak

Institutions: Howard Hughes Medical Institute, Massachusetts General Hospital, Scripps Research Institute

Language: English

Topic: RNA and protein synthesis mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: Howard Hughes Medical Institute, National Institutes of Health, National Institute of General Medical Sciences

Awards: G2911387353, G3201025820, SOLUTION STRUCTURE AND MECHANISM OF A RIBOZYME