The selection of a hydrophobic 7-phenylbutyl-7-deazaadenine-modified DNA aptamer with high binding affinity for the Heat Shock Protein 70

Abstract

Nucleic acids aptamers often fail to efficiently target some proteins because of the hydrophilic character of the natural nucleotides. Here we present hydrophobic 7-phenylbutyl-7-deaadenine-modified DNA aptamers against the Heat Shock Protein 70 that were selected via PEX and magnetic bead-based SELEX. After 9 rounds of selection, the pool was sequenced and a number of candidates were identified. Following initial screening, two modified aptamers were chemically synthesised in-house and their binding affinity analysed by two methods, bio-layer interferometry and fluorescent-plate-based binding assay. The binding affinities of the modified aptamers were compared with that of their natural counterparts. The resulting modified aptamers bound with higher affinity (low nanomolar range) to the Hsp70 than their natural sequence (>5 µM) and hence have potential for applications and further development towards Hsp70 diagnostics or even therapeutics.

Functionally Enhanced XNA Aptamers Discovered by Parallelized Library Screening

“…To investigate the feasibility of converting in vitro selection into a high-throughput screen, we considered the potential for function-enhancing side chains to increase the abundance of high-activity sequences in a random-sequence library (Figure a). We were inspired by the impact that base-modified libraries have made on the results of aptamer selections. − In a large systematic study performed across a range of protein targets, it was discovered that aromatic side chains increased the success rate of aptamer selections from 30 to 84%, with 55% of the selections producing aptamers with a solution binding affinity constant ( K D ) of <1 nM . This finding is consistent with the ability of base-modified aptamers to effectively mimic the paratope surface of an antibody …”

Selection of optimised ligands by fluorescence-activated bead sorting

“…23 The method of modifying nucleic acids reported here has produced aptamers in the range of 1–3 nM, and gave an improvement in affinity of up to 3.3-fold, outperforming even recent de novo -selected uniformly modified aptamers. 59–62 The experimental validation using the EGFR binding assay has been backed up with computational modelling, which provides a structural basis for the changes in affinity and could be used to further refine the aptamers. A typical SELEX process takes 2–3 months for selection and sequence identification.…”

Increasing the functional density of threose nucleic acid

“…19–22 Functionally enhanced libraries prepared using nucleoside triphosphates that are modified to include additional functionality at the C-5 position of pyrimidines and N-7 or C-8 positions of purines offer a different path for improving aptamer activity. 14,23–27 Slow off-rate modified aptamers (SOMAmers), for example, utilize a highly versatile palladium-catalyzed carboxyamidation reaction to install diversity enhancing functional groups at the C-5 position of pyrimidines. 28 This approach revolutionized the field of aptamer-based diagnostics by increasing the success rate of aptamer selections from ∼30% for standard base libraries to 84% for functionally enhanced libraries.…”