Characterizing Aptamers with Reconfigurable Chiral Plasmonic Assemblies

Abstract

Aptamers have emerged as versatile affinity ligands and as promising alternatives to protein antibodies. However, the inconsistency in the reported affinities and specificities of aptamers has greatly hindered the development of aptamer-based applications. Herein, we present a strategy to characterize aptamers by using DNA origami-based chiral plasmonic assemblies as reporters and establishing a competitive hybridization reaction-based thermodynamic model. We demonstrate the characterization of several DNA aptamers, including aptamers for small molecules and macromolecules, as well as aptamers with high and low affinities. The presented characterization scheme can be readily adapted to a wide selection of aptamers. We anticipate that our approach will advance the development of aptamer-based applications by enabling reliable and reproducible characterization of aptamers.

Figure 1

(A) Schematics of DNA origami-based chiral plasmonic probes. The aptamer and a partially complementary strand are incorporated into the probe as an analyte responsive lock. The hybridized state of the two strands corresponds to the closed configuration of the probe (I); the separation of the strands results in open probe configurations (II, III). (B) Configurations of aptamer locks with varied lengths of hybridization (n) between the aptamer and the complementary strand. The hybridization length of n = 0 bp corresponds to the open lock. Increasing n shifts the equilibrium toward the hybridized state of the lock and the closed configuration of the chiral probes. (C) Description of the system in terms of chemical reactions: A, B, and C are the aptamer, analyte, and complementary strand, respectively; ΔG° and K_D are the corresponding Gibbs free energy and dissociation constants, respectively, with K_D2 being the aptamer–analyte dissociation constant of interest. (D) Brief overview of data analysis for the determination of K_D2. The probes with the aptamer and partially complementary strands of different lengths are incubated with or without the analyte. With the parameters obtained from the calibration experiments and computational prediction tools, K_D2 is calculated by measuring the amplitude changes of the CD spectra in the presence and absence of the analyte.

Figure 2

(A) To obtain the coefficient ε between the predicted (ΔG_theory^°) and real (ΔG_r^°) Gibbs free energies, the hybridization length (n) between the aptamer and the complementary strand is varied from 8 to 14 bp (n = {8–14}). (B) Normalized CD spectra of the probes with different hybridization lengths (n). (C) Dependence of ΔG_theory^°(n) on the normalized CD signal at 620 nm is used to calculate ε. (D) To obtain the local concentration of A and C strands (a₀), n is varied between 10 and 13 bp (n = {10–13}). A competitor DNA strand (10 nt) is used as an analyte. (E) By varying n, the dependence of β on K_D1 is used to calculate a₀.

Figure 3

(A) Hybridization of complementary strands to the 5′ or 3′ end of the ATP aptamer with hybridization lengths varied between 9 and 12 bp (n = {9–12}). (B,C) Dependence of β_n on K_D1(n) with the complementary strand hybridizing at the 5′ (B) or 3′ (C) end of the ATP aptamer. K_D2^ATP values of 5.71 ± 0.865 μM (B) and 117 ± 62.8 μM (C) were obtained from the fitting. (D) Dependence of β on the ATP concentration (b₀) produced K_D2^ATP values of 4.54 ± 0.580 μM. (E) Specificity characterization of the ATP aptamer (see Figure S12 for CD spectra).

Figure 4

(A,B) Dependence of β_n on K_D1(n) with complementary strand hybridizing at the 5′ (A) or 3′ (B) of the glucose aptamer.K_D2^glu values of 5.57 ± 0.436 mM (A) and 110 ± 20.0 mM (B) were obtained from the fitting. (C) Specificity characterization of the glucose aptamer (see Figure S13 for CD spectra). (D,E) Dependence of β_n on K_D1(n) with the complementary strand hybridizing at 5′ (D) or 3′ (E) of the thrombin aptamer.K_D2^thr values of 235 ± 24.1 nM (D) and 46.7 ± 7.27 nM (E) were obtained from the fitting. (F) Specificity characterization of the thrombin aptamer (see Figure S14 for CD spectra).