Rational truncation of aptamer for cross-species application to detect krait envenomation

Abstract

In majority of snakebite cases, the snake responsible for the bite remains unidentified. The traditional snakebite diagnostics method relies upon clinical symptoms and blood coagulation assays that do not provide accurate diagnosis which is important for epidemiological as well as diagnostics point of view. On the other hand, high batch-to-batch variations in antibody performance limit its application for diagnostic assays. In recent years, nucleic acid aptamers have emerged as a strong chemical rival of antibodies due to several obvious advantages, including but not limited to in vitro generation, synthetic nature, ease of functionalization, high stability and adaptability to various diagnostic formats. In the current study, we have rationally truncated an aptamer developed for α-Toxin of Bungarus multicinctus and demonstrated its utility for the detection of venom of Bungarus caeruleus. The truncated aptamer α-Tox-T2 (26mer) is found to have greater affinity than its 40-mer parent counterpart α-Tox-FL. The truncated aptamers are characterized and compared with parent aptamer for their binding, selectivity, affinity, alteration in secondary structure and limit of detection. Altogether, our findings establish the cross-species application of a DNA aptamer generated for α-Toxin of Bungarus multicinctus (a snake found in Taiwan and China) for the reliable detection of venom of Bungarus caeruleus (a snake found in the Indian subcontinent).

Figure 1

(A) Secondary structure of α-Tox-FL, α-Tox-T1 and α-Tox-T2 aptamers as obtained by Mfold. Red dotted line represents point of truncation. (B) Showing a comparison of binding of α-Tox-FL, α-Tox-T1 and α-Tox-T2 aptamers to B. caeruleus venom by ELAA.

Figure 2

(A) Showing ELAA results representing selectivity of α-Tox-FL, α-Tox-T1 and α-Tox-T2 aptamers to B. caeruleus venom. (B) Selectivity of α-Tox-FL, α-Tox-T1 and α-Tox-T2 aptamers to B. caeruleus venom is represented as three-colour gradient heat map (a heat-map representation of ELAA response). Red colour indicates highest binding while blue represent the lowest binding.

Figure 3

Circular dichroism (CD) spectrum of α-Tox-FL (A), α-Tox-T1 (B) and α-Tox-T2 (C) aptamers. CD spectra indicate typical B-type stem-loop structure of aptamer that remain unaltered even after truncation.

Figure 4

Apparent dissociation constant curve derived through non-linear regression representing binding affinity (K_d) of α-Tox-FL (A), α-Tox-T1 (B) and α-Tox-T2 (C) aptamers.

Figure 5

Low end detection limit of α-Tox-FL and α-Tox-T2 aptamers. α-Tox-T2 represents more sensitive detection of B. caeruleus venom in comparison to its parent counterpart.

Figure 6

Binding of aptamer to geographically distinct venom of B. caeruleus and other members of ‘BIG Four’ group. KV in parentheses represents venom procured from KV Institute Uttar Pradesh India while P in parentheses represent venom obtained from Premium Serums and Vaccines Pvt.Ltd. Pune, Maharashtra, India.