Exploring the Utility of ssDNA Aptamers Directed against Snake Venom Toxins as New Therapeutics for Snakebite Envenoming

Abstract

Snakebite is a neglected tropical disease that causes considerable death and disability in the tropical world. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapy for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. In this study, we sought to explore the potential of ssDNA aptamers as toxin-specific inhibitory alternatives to antibodies. As a proof of principle model, we selected snake venom serine protease toxins, which are responsible for contributing to venom-induced coagulopathy following snakebite envenoming, as our target. Using SELEX technology, we selected ssDNA aptamers against recombinantly expressed versions of the fibrinogenolytic SVSPs ancrod from the venom of C. rhodostoma and batroxobin from B. atrox. From the resulting pool of specific ssDNA aptamers directed against each target, we identified candidates that exhibited low nanomolar binding affinities to their targets. Downstream aptamer-linked immobilised sorbent assay, fibrinogenolysis, and coagulation profiling experiments demonstrated that the candidate aptamers were able to recognise native and recombinant SVSP toxins and inhibit the toxin- and venom-induced prolongation of plasma clotting times and the consumption of fibrinogen, with inhibitory potencies highly comparable to commercial polyvalent antivenoms. Our findings demonstrate that rationally selected toxin-specific aptamers can exhibit broad in vitro cross-reactivity against toxin isoforms found in different snake venoms and are capable of inhibiting toxins in pathologically relevant in vitro and ex vivo models of venom activity. These data highlight the potential utility of ssDNA aptamers as novel toxin-inhibiting therapeutics of value for tackling snakebite envenoming.

Keywords: SELEX selection; in vitro assays; recombinant toxins; snake venom serine proteases; snakebite envenoming; ssDNA aptamers.

Figure 1

Sequential SELEX selection of aptamers against the recombinant SVSP toxins ancrod and batroxobin. (A,B) DNA was amplified by PCR for each round of toxin selection employed (Lane 1–14; size, DNA ≈ 104 bp), with non-specific bound ssDNA eliminated by counter-selection (CS, highlighted by arrow) employed at round 12. Data for ancrod are shown in (A) and batroxobin in (B). (C,D) The fluorescence intensity of the eluted ssDNA solution from each round of selection was measured at an excitation wavelength of 470 nm and an emission of 515 nm. Error bars represent the standard error (SEM) of triplicate measurements, with data for ancrod shown in (C) and batroxobin in (D). (E,F) Colony PCR was performed by ligation into a cloning vector and transformation into E. coli competent cells, with validation performed on ten random colonies to demonstrate that the inserted DNA (Lane 1–10; size ≈ 300 bp) was successfully ligated into the pCR2.1-TOPO cloning vector (Lane 11; size ≈ 3956 bp). Data for ancrod are shown in (E) and batroxobin in (F).

Figure 2

The equilibrium K_ds of the highest affinity ssDNA aptamers were derived from the fluorescence binding curves for each toxin target. The data shown represent the highest affinity candidate aptamers for ancrod (ancrod-7, ancrod-11 and ancrod-55) (A) and batroxobin (batroxobin-5, batroxobin-26 and batroxobin-29) (B). Concentration-dependent data were generated at an excitation wavelength of 470 nm and an emission of 515 nm, and K_ds were calculated via nonlinear regression analysis of the resulting saturation curves. Data represent means of duplicate measurements, and error bars represent standard deviations (SD).

Figure 3

Quantification of binding levels between the selected ssDNA aptamers (ancrod-55 and batroxobin-26) and the SVSP recombinant toxins (ancrod and batroxobin) and a panel of native snake venoms by ALISA. Data are shown for the aptamers ancrod-55 (A) and batroxobin-26 (B). The data on the left show the binding levels obtained against the two recombinant toxins that the aptamers were selected against and the corresponding snake venoms that these toxins are derived from. The data on the right show binding levels obtained against a broad panel of 11 distinct snake venoms. All data represent the mean of duplicates measured at 450 nm, and error bars represent the standard deviation (SD) of the duplicate measurements.

Figure 4

SDS-PAGE gel electrophoresis visualisation of fibrinogenolysis demonstrates that ssDNA aptamers directed against ancrod and batroxobin inhibit the fibrinogenolytic activity of recombinant toxins and the corresponding snake venoms. The fibrinogenolytic activity of: (A) ancrod and inhibition by the aptamer ancrod-55, (B) batroxobin and inhibition by batroxobin-26, (C) C. rhodostoma venom and inhibition by ancrod-55, and (D) B. atrox venom and inhibition by batroxobin-26. The figure shows a reduced 8% SDS-PAGE, visualised by Coomassie blue staining. All panels have the following layout: Lane 1, protein marker (PM); Lane 2, human plasma fibrinogen; Lane 3, toxin (ancrod or batroxobin) or venom (C. rhodostoma or B. atrox) + fibrinogen; Lane 4, toxin or venom + fibrinogen + aptamer (ancrod-55 or batroxobin-26, 1 pM); Lane 5, toxin or venom only.

Figure 5

Fibrinogen consumption and prolongation of the PT and aPTT induced by recombinant toxins and corresponding snake venoms are reduced by the ssDNA aptamers in diluted citrated human PPP samples. (A) Fibrinogen concentrations were quantified using an excess of thrombin to convert fibrinogen to fibrin, (B) PT, which measures the combined effect of clotting factors of the extrinsic and common coagulation pathways (in seconds), and (C) aPTT, which measures the combined effect of the clotting factors of the intrinsic and common coagulation pathways. PPP samples were spiked with 0.6 ng of either recombinant toxin or native venom only (i.e., ancrod, batroxobin, C. rhodostoma or B. atrox); toxin/venom + 1 pM of specific aptamer (ancrod/C. rhodostoma with ancrod-55, batroxobin/B. atrox with batroxobin-26); or toxin/venom + 0.5 µg of specific commercial antivenom (ancrod/C. rhodostoma with Malayan pit viper antivenom, batroxobin/B. atrox with SORO antibotropico/crotalico antivenom). Controls consisted of aptamer-only samples, antivenom-only samples, and saline solution (negative control). Error bars represent the standard deviation (SD) of duplicate measurements.