Directed Evolution of 2′-Fluoro-Modified, RNA-Supported Carbohydrate Clusters That Bind Tightly to HIV Antibody 2G12

Abstract

Carbohydrate binding proteins (CBPs) are attractive targets in medicine and biology. Multivalency, with several glycans binding to several binding pockets in the CBP, is important for high-affinity interactions. Herein, we describe a novel platform for design of multivalent carbohydrate cluster ligands by directed evolution, in which serum-stable 2′-fluoro modified RNA (F-RNA) backbones evolve to present the glycan in optimal clusters. We have validated this method by the selection of oligomannose (Man9) glycan clusters from a sequence pool of ∼1013 that bind to broadly neutralizing HIV antibody 2G12 with 13 to 36 nM affinities.

From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma

“…Open and was used to synthesize TNA containing functionalized nucleobases 68 . In another example, T7 RNAP mutant Y639F was shown to be efficient for the transcription of RNA with 2'-deoxy-2'-fluoro (5-ethynyl) uridine triphosphate and other natural NTPs, and used for the evolution of 2'-F-modified RNA-scaffolded carbohydrate clusters 67,168 . Very recently, Niu and co-workers synthesized C8-alkyne-FANA UTP, and demonstrated its enzymatic incorporation into FANA by Tgo DNAP 181 .This work further enriched the XNA toolbox with components containing clickable handles.…”

“…Introduction of two or more modifications into different moieties of one nucleic acid at the same time is supposed to introduce multiple changes in properties or functions into this nucleic acid, which further expands the chemical diversity of nucleic acids, and thus is attractive. For example, 2'-deoxy-2'-fluoro (5-ethynyl) uridine triphosphate was synthesized and employed to introduce both physiological stability and handles to attach functionalities, such as carbohydrates in this reported case, onto the nucleobases into the same RNA scaffold67 . In another example, Chaput and coworkers reported the synthesis and polymerase recognition of α-L-threofuranosyl uridine nucleoside triphosphate (tUTP) analogs with chemically diverse functional groups attached to C-5 position, which are very useful for the production or evolution of highly functionalized TNA 68 .…”

“Click handle”-modified 2′-deoxy-2′-fluoroarabino nucleic acid as a synthetic genetic polymer capable of post-polymerization functionalization

“…Carbohydrate-modied nucleic acids have emerged as a promising technology to develop affinity reagents for glycan-binding proteins (GBPs) and vaccine epitopes. 33,37 To demonstrate that cmFANA can serve as a general platform for carbohydrate-modied XNAs, we investigated the click conjugation of azidofunctionalized glycans with cmFANA (Fig. 5a).…”

“…On the other hand, installing functional groups to the nucleobases has been demonstrated to be an effective strategy to expand the chemical repertoire and enhance the bioactive functions in natural nucleic acids. [24][25][26][27] An array of hydrophobic groups have been incorporated in both DNA and RNA aptamers, with notable examples including slow off-rate modied aptamers (SOMAmers) 28 and highly functionalized nucleic acid polymers (HFNAPs). 29 Impressively, in 2018 Chaput and coworkers reported a TNA aptamer uniformly incorporating a 7deaza-7-phenyl guanidine against HIV reverse transcriptase with nanomolar affinity.…”

Probing Multivalent Carbohydrate-Protein Interactions With On-Chip Synthesized Glycopeptides Using Different Functionalized Surfaces

“…Therefore, various studies in the last years focused on the optimum glycan presentation, concentration, flexibility, orientation, and density in the array format (Oyelaran et al, 2009;Müller et al, 2016;Kim et al, 2018;Mende et al, 2019;Valles et al, 2019;Di Maio et al, 2021). In addition, a plethora of multivalent glycan scaffolds have been investigated with diverse size and shape to mimic the natural recognition (Cecioni et al, 2015;Delbianco et al, 2016;Redman and Krauss, 2021). Peptide scaffolds have been widely studied due to their simple synthesis via solid phase peptide synthesis, (Merrifield, 1963) offering well-defined monodisperse structures.…”