Selection of DNA Aptamers with Binding Affinity to Human Norovirus

Introduction: Human noroviruses (HuNoV) are the leading cause of foodborne disease.  Currently, there is no easy way to detect HuNoV contamination.  A major reason for this is the so-called “needle in a haystack” dilemma, meaning that it is necessary to concentrate and purify small numbers of viruses from the sample matrix prior to the application of molecular-based detection.  Aptamers [small, single-stranded (ss) DNA or RNA molecules that naturally fold into complex three-dimensional shapes] are emerging ligands for pathogen capture.  These demonstrate advantages over traditional capture ligands like antibodies, including reduced cost, ease of production and modification, and improved stability. 

Purpose: To develop and characterize ssDNA aptamers with binding specificity to the HuNoV GII.2 strain Snow Mountain virus (SMV). 

Methods: Aptamers were selected from a large combinatorial library of random ssDNA molecules using the SELEX (Systematic Evolution of Ligands by Selective Enrichment) method targeting purified, intact virus.  After multiple rounds of SELEX (and counter-selection), enriched aptamer pools were cloned, sequenced and their secondary structure analyzed using DNA Mfold.  Preliminary data on their binding affinity was determined using an Enzyme-Linked Aptamer (ELA) assay developed as part of this work.

Results:  Thirty-eight aptamer candidates were identified.  Of these, five aptamer sequences (SMV-5, SMV-18, SMV-22, SMV-21S9, SMV-22S9) were most predominant, represented from 2-5 times in each purified aptamer pool.  Sequence analysis of the aptamers revealed only a few common domains but similarity in predicted structural folding, with all aptamers having a dominant loop and three protruding hairpins. Equilibrium dissociation constants (K_d), a measure of binding affinity, were 0.04, 0.045, 0.142, 0.05, 0.016, µM for aptamers SMV-5, SMV-18, SM-22, SMV-21S9, and SMV-22S9, respectively. 

Significance: Aptamers with binding affinity to SMV are promising ligands for pathogen capture prior to molecular detection.  Future studies focus on using these to develop practical, inexpensive approaches to concentrate HuNoV from foods and environmental samples.