Nucleic Acid Therapeutics, 2011, Vol. 21, No. 4, pp 253-263.
Nucleotide bias observed with a short SELEX RNA aptamer library
William H. Thiel, Thomas Bair, Kristina Wyatt Thiel, Justin P. Dassie, William M. Rockey, Craig A. Howell, Xiuying Y. Liu,
Adam J. Dupuy, Lingyan Huang, Richard Owczarzy, Mark A. Behlke, James O. McNamara II, and Paloma H. Giangrande
Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful in vitro selection process used for over 2 decades
to identify oligonucleotide sequences (aptamers) with desired properties (usually high affinity for a protein target) from randomized
nucleic acid libraries. In the case of RNA aptamers, several highly complex RNA libraries have been described with RNA sequences ranging
from 71 to 81 nucleotides (nt) in length. In this study, we used high-throughput sequencing combined with bioinformatics analysis to
thoroughly examine the nucleotide composition of the sequence pools derived from several selections that employed an RNA library (Sel2N20)
with an abbreviated variable region. The Sel2N20 yields RNAs 51 nt in length, which unlike longer RNAs, are more amenable to
large-scale chemical synthesis for therapeutic development. Our analysis revealed a consistent and early bias against inclusion of adenine,
resulting in aptamers with lower predicted minimum free energies (ΔG) (higher structural stability). This bias was also observed in
control, "nontargeted" selections in which the partition step (against the target) was omitted, suggesting that the bias occurred in 1 or
more of the amplification and propagation steps of the SELEX process.
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