In vitro Selection of High Affinity DNA and RNA Aptamers that Detect Hepatitis C Virus Core Protein of Genotypes 1 to...

Hepatitis C virus (HCV) core is a highly conserved and multifunctional protein that forms the viral capsid, making it an attractive target for HCV detection and inhibition. Aptamers are in vitro selected, single-stranded nucleic acids (RNA or ssDNA) with growing applicability in viral diagnostics and therapy. We have carried out DNA and RNA in vitro selection against six different variants of HCV core protein: two versions of the full-length protein of genotype 1, and the hydrophilic domain of genotypes 1 to 4. The aptamer populations obtained were analyzed by means of Ultra-Deep Sequencing (UDS), the most abundant sequences were identified and a number of highly represented sequence motifs were unveiled. Affinity (measured as the dissociation constant, Kd) of the most abundant DNA and RNA aptamers were quantified using Enzyme-Linked OligoNucleotide Assay (ELONA)-based methods. Some aptamers with nanomolar or subnanomolar Kd values (as low as 0.4 nM) were the common outcome of DNA and RNA selections against different HCV core variants. They were tested in sandwich and competitive biosensor assays, reaching a limit of detection for HCV core of 2 pM. Additionally, the two most prevalent and high affinity aptamers were assayed in Huh-7.5 reporter cell lines infected with HCV, where they decreased both the viral progeny titer and the extracellular viral RNA level, while increasing the amount of intracellular viral RNA. Our results suggest that these aptamers inhibit HCV capsid assembly and virion formation, thus making them good candidate molecules for the design of novel therapeutic approaches for hepatitis C.