Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer

Abstract

DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson-Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.

Keywords: SELEX; X-ray crystallography; malaria diagnosis; point-of-care diagnostics.

Fig. 1.

The tetrameric PfLDH binds two DNA aptamers. (A) Crystal structure of tetrameric PfLDH in complex with two 2008s DNA aptamers. The A, B, C, and D subunits of PfLDH are colored in white, yellow, red, and pink, respectively. One 2008s aptamer (designated aptamer A) spans subunits A and B, whereas the other 2008s aptamer (designated aptamer B) spans subunits C and D. (B) Structure and sequence of the 2008s DNA aptamer alone in the complexed state. The 2008s aptamer shows a distorted hairpin comprising a B-helical stem, an asymmetric internal loop containing flipped base T⁹, and an apical loop containing flipped base A¹⁶. Clear electron density was observed in the crystal structure for the first 27 bases of each aptamer (colored in black in sequence), and no electron density was observed for the last eight bases at the 3′ of each aptamer (colored in gray in sequence). (C) Isothermal titration calorimetry titrations for 2008s aptamer binding to PfLDH (Left) and human LDHA1 and LDHB (Right). Data were fitted to a single-site model revealing K_D = 42 nM and approximate 2:1 protein subunit:aptamer molar stoichiometry (Lower Left).

Fig. 2.

The substrate specificity loop at the protein:aptamer binding interface determines the discrimination of the aptamer for PfLDH over human LDH. (A) Direct interactions of the 2008s DNA aptamer with PfLDH. Extensive interactions are observed between bases 6–11 and base 22 of the 2008s aptamer with the substrate specificity loop in subunit A/C (colored in white), and less extensive interactions are observed between bases 17 and 19 and subunit B/D (colored in yellow). Each aptamer buries 895 Å on subunit A/C and buries 394 Å on subunit B/D. (B) Discrimination of aptamer specificity in binding to PfLDH over hLDH is explained by differences in the substrate specificity loop. The substrate specificity loops of PfLDH and hLDHB are colored red and blue, respectively.

Fig. 3.

Visualization of aptamer and substrate binding sites on PfLDH. (A) Substrate-bound PfLDH (PDB ID code: 1LDG; ref. 21) was superposed onto subunits A and B of aptamer-bound PfLDH tetramer. For clarity, only the superposed oxamate (in dark pink and magenta) and NAD⁺ (in slate and dark blue) from substrate-bound PfLDH were shown as space-filled models. (B) As seen from the structure of the substrate-bound PfLDH, when substrate and cofactor are bound, the substrate-specificity loop of PfLDH (shown in pale green) closes over the active site covering the bound substrate and cofactor. (C) When 2008s DNA aptamer is bound to PfLDH, the substrate-specificity loop (shown in pink) is held by the aptamer in an open conformation, exposing the active site. Only the superposed oxamate and NAD⁺ from the substrate-bound PfLDH are shown as ball-and-stick models for clarity.

Fig. 4.

Aptamers conjugated to gold nanoparticles to demonstrate potential for application. (A) Principle of approach. (B) Transmission electron microscopy images of nonconjugated gold nanoparticles (Left), aptamer-conjugated gold nanoparticles (Center), and aptamer-conjugated gold nanoparticles in presence of PfLDH (Right). (C) Qualitative observation using gold nanoparticles to detect PfLDH specifically at 25 ng/µL. (D) Quantitative absorbance of data in C.