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.

Fig. 1. Synthesis of PhBu-modified dA^*TP and phosphoramidite 7.

Reagents and conditions A: (i) but-3-yn-1-ylbenzene (10 equiv.), Pd(OAc)₂ (0.1 equiv.), CuI (0.1 equiv.), TPPTS (0.1 equiv.), TEA (6 equiv.), MeCN/H₂O (1:1), RT, Ar, overnight; (ii) H₂ (baloon), 10% Pd/C (0.1 equiv.), MeOH, reflux, overnight; iii) 1. POCl₃ (1.2 equiv.), PO(OMe₃)₃, 0 °C, 2 h; 2. (NHBu₃)₂H₂P₂O₇ (5 equiv.), Bu₃N (4 equiv.), DMF (dry), 0 °C, 1 h; 3. 2 M TEAB; Reagents and conditions B: iv) DMTrCl (1.2 equiv.), DMAP (0.1 equiv.), pyridine (dry), RT, 6 h; v) N,N-dimethylformamide dimethylacetal (14 equiv.), DMF (dry), 40 °C, Ar, 4 h; vi) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.2 equiv.), DIPEA (2.5 equiv.), DCM (dry), 0 °C to RT, 1.5 h.

Fig. 2. Scheme of the PEX construction of modified DNA and denaturing PAGE analysis of PEX reaction with DNA random library using KOD XL DNA polymerase.

Scheme of the PEX (A); Denaturing PAGE analysis (B): Lane 1 (P): primer, Lane 2 (Nat): DNA library amplified by natural dNTPs, Lane 3 (Mod): DNA library amplified with modified dA*TP and natural dCTP, dGTP, dTTP, Lane 4 (Pool): DNA library scaled-up for generation of modified starting library for SELEX, Lane 5 (L): FAM-labelled ssDNA ladder.

Fig. 3. Principle of the modified SELEX procedure used in this study (scheme modified from ref. ).

a The initial modified library is generated by PEX from 5′-biotinylated ssDNA library followed by b single stranded generation via magneto-separation. c A counter-selection step against the beads was carried out prior to d positive selection with the target. e, f The bound ssDNA is separated from the non-binders by washing and g the released ssDNA is amplified by PCR using biotinylated forward primer and phosporylated reverse primer, which allows for h subsequent ssDNA generation via Lambda exonuclease digestion. i The resulting 5′-biotinylated ssDNA is ready for PEX and another round of modified SELEX.

Fig. 4. Binding affinity results of the natural HSNat-2 sequence and modified HSc-2 aptamer.

a, b the potential predicted secondary structure for the HS-2 aptamers as determined by MFold. BuPh-modifed dA* side chains are highlighted in red for visualisation. c, d The corresponding binding curves for HS-2 aptamers as determined by BLI, different aptamer concentrations were used as analyte and the steady state binding affinity [K_d] was calculated. e, f The corresponding binding curves for HS-2 aptamers as determined by the fluorescent-plate-based binding assay. The raw fluorescent data were exported into Graph Prism and the binding affinity [K_d] of each aptamer calculated. Error bars represent the average values of 2 independent experiments.

Fig. 5. Binding affinity results of the natural HSNat-9 sequence, modified HSc-9 aptamer and the modified truncated HSc-9.1 aptamer.

a–c The potential predicted secondary structures for the HS-9 aptamers as determined by MFold. PhBu-modifed dA* bases with side chains are highlighted in red for visualisation. d–f The corresponding binding curves for HS-9 aptamers as determined by BLI, different aptamer concentrations were used as analyte and the steady state binding affinity [K_d] was calculated. g–i The corresponding binding curves for HS-9 aptamers as determined by the fluorescent-plate-based binding assay. The raw fluorescent data were exported into Graph Prism and the binding affinity [K_d] of each aptamer calulated. Error bars represent the average values of two independent experiments.