Methods for Directed Evolution of Carbohydrate Clusters on 2���-fluoro-modified RNA

Forty years after the discovery of HIV and after multiple vaccine efficacy trials, no HIV-1 candidate vaccine has proven effective enough for clinical use. One of the major reasons that HIV-1 has proven to be such a challenging target for vaccine design is that the surface of the viral envelope spike protein (Env) is protected by extensive glycosylation. The dense glycosylation paired with high sequence variability of the virus makes it difficult for the immune system to generate broadly neutralizing antibodies (bnAbs) against the polypeptide surfaces of Env. However, up to 20% of patients with HIV do eventually develop bnAbs, many of which bind to epitopes comprising glycans or glycans and conserved protein residues. Significant interest lies in the development of structures able to mimic bnAb epitopes. Here we work to develop a method called Capture-SELMA, for the directed evolution of serum-stable, modified-RNA carbohydrate clusters able to elicit production of bnAbs towards HIV. In Capture-SELMA, or Capture-SELection of Modified Aptamers, carbohydrate-modified RNAs bearing fluoro-modified sugar backbones to impart serum stability, are noncovalently tagged with their encoding DNAs through a DNA ���capture strand���. We have validated this method by the selection of oligomannose (Man9) glycan clusters from a sequence pool of ���10^13 that bind to broadly neutralizing HIV antibody 2G12 with 13 to 36 nM affinities. An alternative platform, Hairpin-SELMA has also been developed to address some discovered Capture-SELMA limitations and will continue to be investigated. This method builds off our lab���s initial SELMA platform for evolving DNA-supported glycoclusters, introducing customizability to the backbone, in the form of 2���-modifications, with the goal of evolving glycoclusters with superior serum stability.