Self-folded Primers as an Alternative to Single-stranded Primers in an Oligonucleotide Library Designed for Aptamer Screening

Document Type : Research Paper


Department of Chemistry, Marshall University, Huntington, WV 25755, United States


In recent years, oligonucleotide libraries have been used to screen for high-affinity and high-selectivity aptamers that target proteins, peptides, small molecules, or cells. The members of a traditional oligonucleotide library contain a randomized central region with a single-stranded primer region at each end for amplifying target-bound central sequences. However, the single-stranded primers may interact with complementary sequences in the central region of the oligonucleotide and interfere with target-binding. The authors proposed a new library design that uses self-folded motifs to replace the single-stranded primers. For proof of concept, highly structured intercalated motifs (i-motifs) were used to construct the primer regions, and DNA oligonucleotides containing a known streptavidin-binding aptamer in the central region were used to evaluate the performance of the i-motif primers. The applicability of i-motif primers for polymerase chain reaction (PCR) was evaluated as well. The experimental results indicate that the self-folded i-motif primers do not interrupt streptavidin binding to the central region of the DNA oligonucleotide, and that they are PCR-friendly. Finally, when these self-folding primers were used to construct the members of an otherwise traditional DNA library, the target molecules were successfully collected, demonstrating that such primers are a viable alternative to the traditional single-stranded primers in the members of an oligonucleotide library used for aptamer screening.