Rational design of structurally rich DNA aptamer screening libraries guided by empirical sequence trends and stable...

Aptamers are typically identified by screening combinatorial libraries with random sequence populations. Structured oligonucleotide libraries which include self-folded motifs such as canonical hairpins or noncanonical G quadruplexes offer potential advantages in aptamer screening by increasing conformational complexity to promote target binding and selection efficiency. To explore these advantages, DNA libraries were rationally designed by integrating empirical sequence biases and experimentally validated thermodynamic motifs as follows: (1) an empirical library enriched in guanine and thymine, reflecting nucleotide composition patterns observed in database aptamer sequence entries; (2) a tetraloop library, embedded with known DNA hairpin motifs with high thermodynamic stability; and (3) a standard library composed of conventional random oligonucleotide sequences. Minimum free energy calculations by mFold predicted a hierarchy of thermal stabilities with the tetraloop library exhibiting the highest stability followed by the standard, then empirical libraries. Experimental validation via UV-vis melting analysis confirmed this predicted trend in thermal stability. Additionally, quantitative polymerase chain reaction (qPCR) analysis demonstrated efficient and unbiased amplification efficiency across all three libraries. Finally, an adapted next generation sequencing (NGS) protocol enabled sequence analysis that confirmed compatibility of each library with high throughput Illumina platforms. By biasing heterogeneous DNA populations towards empirical nucleotide compositions and experimentally validated structural elements, this approach enables the design of oligonucleotide screening libraries to enrich potential target-binding motifs.