Virus–based SELEX (viro-SELEX) allows development of aptamers targeting knotty proteins.

Abstract

Nucleic acid aptamers, often termed chemical antibodies, are short, single-stranded DNA or RNA molecules (20–100 nucleotides in length) with defined structures that can specifically bind to a molecular target via three-dimensional structures. Similarly to the way antibodies bind to antigens, aptamers specifically recognize and bind to their cognate targets through unique threedimensional structures. SELEX (systematic evolution of ligands by exponential enrichment) is a gold-standard methodology for generating aptamers, in which an iterative selection procedure — including binding, partitioning, recovery and re-amplification steps — is conducted. Specific sequences (that is, aptamers) can be enriched and dominate the population of library species. In this study, we developed a novel SELEX using surrogate baculoviruses which gives us an edge over the existing protein-based SELEX methods in that, it can avoid the false-positive aptamers that bind to the portions of the protein that ideally, in a real life scenario is not exposed at all. To perform this, we prepared all the raw materials necessary for example, recombinant proteins, and surrogate baculoviruses expressing our protein of interest (in this case hemagglutinin of the influenza A virus H1N1 subtype). We performed SELEX with the above raw material with increasing stringencies in every round and shortlisted the scaffolds that gave good binding to the proteins and viruses. These aptamers were tested for binding purposes and competition and were modified to make them fit into the lateral flow assays. As the simple gold nanoparticle assays were not great enough for the real life detection limits, we improvised the system using fluorescent dyes on the aptamers which gave at least 10-12 fold better sensitivity in terms of detection limits as compared to that of the gold nanoparticle based lateral flow assay system. In summary, we devised a novel virus-based SELEX using baculovirus and also proved that using surrogate baculoviruses indeed helps in better selection and also helps in getting broad selectivity. Acquired aptamers generated from this new method showed high binding affinities and specificities. With this approach, I was able to develop an aptamer-based subtype-specific diagnostic system for influenza virus H1N1. Also by using two different strains of the H1N1 subtype, we could prove that strains with higher percentage of identity can easily be detected by our aptamers and strains with lower identity could be easily excluded. Also, it was of great interest to know that even though one more strain of the H1N1 subtype was not used in the selection process, our aptamers could bind to that. Thus, here in we have given a proof of concept for the viro-SELEX method devised by us in our previous paper.