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

Area of Science:

• Biochemistry
• Molecular Biology
• Bioinformatics

Background:

• Aptamers are typically identified using random sequence libraries.
• Structured oligonucleotide libraries with self-folded motifs can improve aptamer screening efficiency.
• Integrating sequence biases and thermodynamic motifs into library design is a promising approach.

Purpose of the Study:

• To explore the advantages of structured oligonucleotide libraries in aptamer screening.
• To rationally design DNA libraries with empirical sequence biases and validated thermodynamic motifs.
• To assess the thermal stability, amplification efficiency, and sequencing compatibility of designed libraries.

Main Methods:

• Designed three DNA libraries: empirical (Guanine/Thymine enriched), tetraloop (hairpin motifs), and standard (random).
• Utilized mFold for minimum free energy calculations to predict thermal stability.
• Performed UV-vis melting analysis for experimental validation of thermal stability.
• Employed quantitative polymerase chain reaction (qPCR) for amplification efficiency analysis.
• Adapted next-generation sequencing (NGS) protocols for library compatibility assessment.

Main Results:

• Predicted and experimentally validated a hierarchy of thermal stability: tetraloop > standard > empirical libraries.
• Demonstrated efficient and unbiased amplification across all three library types via qPCR.
• Confirmed compatibility of all designed libraries with high-throughput Illumina sequencing platforms.
• Showcased that biasing DNA populations towards specific nucleotide compositions and structural elements enriches potential target-binding motifs.

Conclusions:

• Rationally designed structured oligonucleotide libraries offer advantages for aptamer screening.
• The integration of empirical sequence biases and validated thermodynamic motifs enhances library design.
• This approach facilitates the development of oligonucleotide screening libraries with enriched target-binding potential.