Abstract
Carbohydrate binding proteins (CBPs) are alluring and challenging targets in biology. CBPs achieve high-affinity interactions with their substrates through multivalent interactions with carbohydrates, often in branched, heterogeneous structures. Many cell recognition events and host-pathogen interactions are mediated by these carbohydrate-protein interactions. Therefore, the development of glycan-based mimics and disruptors of these interactions have applications in studying target CBPs, carbohydrate-based vaccines, immunomodulating agents, diagnostics, and therapeutics against cancer and other diseases. Previously, the Krauss lab has developed SELection with Modified Aptamers (SELMA), to evolve nucleic acid-scaffolded glycan clusters to develop high-affinity aptamers towards CBPs. Herein, I describe the extension of Hairpin-SELMA to fully 2′-modified RNA scaffolds, which confer enhanced serum stability, using engineered polymerases (KOD-DGLNK, SFM4-3, and Therminator).
This new SELMA methodology was first applied to HIV broadly neutralizing antibodies (bnAbs) 2G12 and PGT128, which recognize clustered high-mannose (Man9) glycans on the viral envelope protein gp120. Four parallel selections were conducted towards these antibodies with two Man9 glycoconjugates over eleven to twelve rounds of selection. The binding of individual aptamers was characterized on a library scale through a novel high-throughput NGS-based binding assay, estimating the KD of thousands of unique sequences from a single multiplexed experiment. Multiple sub-100 nM KD binding sequences were identified in three of the four libraries with two different types of novel aptamers identified using SELMA methodologies. The first are Man9GlcNAc2 glycoclusters towards the bnAb 2G12 and the second are Man9cyclohexyl glycoclusters towards the bnAb PGT128. Finally, to explore the generality of the 2’-modified RNA SELMA platform beyond HIV antibody targets, we applied it to the chemokine CXCL8 using heparan sulfate-derived glycans and observed target-dependent enrichment across selection rounds. Together, this work establishes 2’-modified RNA and our novel NGS-based high-throughput assay as a generalizable platform for evolving multivalent glycoaptamers against relevant CBPs.