Evoking picomolar binding in RNA by a single phosphorodithioate linkage

Abstract

RNA aptamers are synthetic oligonucleotide-based affinity molecules that utilize unique three-dimensional structures for their affinity and specificity to a target such as a protein. They hold the promise of numerous advantages over biologically produced antibodies; however, the binding affinity and specificity of RNA aptamers are often insufficient for successful implementation in diagnostic assays or as therapeutic agents. Strong binding affinity is important to improve the downstream applications. We report here the use of the phosphorodithioate (PS2) substitution on a single nucleotide of RNA aptamers to dramatically improve target binding affinity by ∼1000-fold (from nanomolar to picomolar). An X-ray co-crystal structure of the α-thrombin:PS2-aptamer complex reveals a localized induced-fit rearrangement of the PS2-containing nucleotide which leads to enhanced target interaction. High-level quantum mechanical calculations for model systems that mimic the PS2 moiety and phenylalanine demonstrate that an edge-on interaction between sulfur and the aromatic ring is quite favorable, and also confirm that the sulfur analogs are much more polarizable than the corresponding phosphates. This favorable interaction involving the sulfur atom is likely even more significant in the full aptamer-protein complexes than in the model systems.

Recent Methods for Purification and Structure Determination of Oligonucleotides

“…With a salt-gradient elution mode on tertiary or quaternary ammonium-derivatized polymeric adsorbent or porous silica, the oligonucleotides could be separated by the ion-exchange chromatography method. Several kinds of advance structure, such as secondary, tertiary, dimers, or aggregates may exist in oligonucleotides, so denaturing conditions are required for these types of structures which could be achieved by adding organic solvents such ethanol or acetonitrile, using high-pH buffers or high temperature (55 to 65 °C depending on the column) [11,14,26]. …”

“…Additionally, some modifications have been introduced, such as phosphorothioate linkages, incorporating modified nucleosides as 2′-OMe, 2′-OMOE, or 2′-F in the sequence, and attaching polyethylene glycol at the terminus to the structures of oligonucleotides in order to increase stability and bioavailability. However, these modifications also enhanced the challenges for analysis and purification [10,11,12]. …”

Origins of the enhanced affinity of RNA–protein interactions triggered by RNA phosphorodithioate backbone modification

“…23 Recent studies have shown that combined 2′-OMe-PS2 or 2′-F-PS2 substitution experiments with in vitro selected RNA aptamers 24, 25 led to either a reduction or increase in binding affinity. 26 A destabilizing effect by 2′-modification could be due to altered sterics (2′-OMe vs. 2′-OH), loss of H-bonding (2′-OMe vs. 2′-OH), or arise as a consequence of modifying a residue that adopts a C2′- endo pucker, i.e. shifting the conformational equilibrium to C3′- endo compared to the native ribose (as seen with the 2′-F analog).…”

“…Evaluation consisted of using a serial dilution of MS2 protein screened against individual hairpin RNA variants that were immobilized onto streptavidin coated BLI sensors. 26, 29 Binding and dissociation rates of native hairpin RNA were determined in parallel to the variants. Supplementary Figure 1 (SF-1) shows the characterization of native RNA and all of its PS2 modified variants.…”

“…The more dramatic increases in binding affinity for certain PO2 → PS2 substitutions are consistent with our recent results based on PS2-modified aptamers targeting VEGF or thrombin. 26 What are the underlying reasons for affinity increases upon PS2 substitution? The fact that the P-S bond is longer than the P-O bond (by ca.…”