Target Affinity and Structural Analysis for a Selection of Norovirus Aptamers

Area of Science:

• Molecular biology
• Biochemistry
• Analytical chemistry

Background:

• Aptamers are single-stranded oligonucleotides with high affinity for specific molecules, used in diagnostics and therapeutics.
• Existing norovirus aptamers lack thorough characterization, hindering the development of norovirus detection assays.

Purpose of the Study:

• To characterize seven known norovirus aptamers for their binding affinity, structural properties, and suitability for norovirus extraction and detection assays.

Main Methods:

• Filter retention assays were used to determine aptamer affinities against various norovirus genotypes (GI.1, GI.7, GII.3, GII.4 New Orleans, GII.4 Sydney) using virus-like particles.
• Circular dichroism (CD) spectroscopy was employed to analyze aptamer structures, including comparisons between biotinylated and non-biotinylated forms.
• Dot-blot and pull-down assays were conducted to demonstrate the feasibility of aptamer-based norovirus assays.

Main Results:

• Four aptamers showed affinity for norovirus GII.4 strains, while three also bound to GII.3 and GI.7 strains. No affinity was observed for GI.1.
• Equilibrium dissociation constants (Kd) for GI.7, GII.3, GII.4 New Orleans, and GII.4 Sydney ranged from 71 ± 38 nM to 1777 ± 1021 nM.
• Six aptamers exhibited a B-DNA structure, and one displayed a G-quadruplex hybrid structure. Biotinylation did not alter aptamer structures.

Conclusions:

• The characterization of these seven norovirus aptamers provides essential data for future development of aptamer-based diagnostic and extraction tools.
• Demonstrated feasibility in dot-blot and pull-down assays supports the potential of these aptamers in norovirus detection systems.
• Further research can build upon this knowledge base for aptamer modification and the creation of novel norovirus assay platforms.