Novel combinatorial selection of phosphorothioate oligonucleotide aptamers

Area of Science:

• Molecular Biology
• Biochemistry
• Immunology

Background:

• Nuclear factor for human IL6 (NF-IL6) is a transcription factor crucial for inflammatory gene induction.
• Existing methods for targeting NF-IL6 face challenges with nuclease resistance and binding specificity.
• Phosphorothioate DNA aptamers offer potential for enhanced stability and targeted therapeutic applications.

Purpose of the Study:

• To develop and screen nuclease-resistant phosphorothioate DNA aptamers for high-affinity binding to NF-IL6.
• To investigate the binding characteristics and stoichiometry of selected aptamers.
• To explore the potential of these aptamers as therapeutic agents targeting inflammatory pathways.

Main Methods:

• A novel combinatorial approach was employed for the construction and screening of DNA aptamers.
• PCR amplification using dNTP(alphaS) was utilized to generate phosphorothioate backbone substitutions.
• A 22-nucleotide-long random duplex library was screened for specific binding to NF-IL6.

Main Results:

• Specific 22-mer thiophosphate backbone substituted aptamers with nanomolar binding affinity for NF-IL6 were selected.
• These aptamers demonstrated a distinct consensus sequence compared to wild-type NF-IL6 binding sites.
• Selected aptamers exhibited a higher binding stoichiometry (2 dimers/duplex) compared to wild-type sequences (1 dimer/duplex).

Conclusions:

• Novel phosphorothioate DNA aptamers targeting NF-IL6 have been successfully developed using a combinatorial approach.
• These aptamers possess enhanced nuclease resistance, specificity, and binding stoichiometry.
• The findings suggest potential therapeutic applications for these aptamers in modulating inflammatory responses.