Researchers from THSTI, AIIMS and IIT Indore have found that the aptamer originally developed against alpha toxin of Bungarus multicinctus krait snake found in China and Taiwan can be used for reliable detection of B. caeruleus krait species found in the Indian subcontinent. The truncated aptamer shows better affinity, sensitivity and binding to the target than the original aptamer.
Researchers have found a DNA aptamer (a small, single-stranded DNA molecule that binds to a specific target molecule) originally developed against alpha toxin of Bungarus multicinctus krait snake can be used for reliable detection of another krait snake species — Bungarus caeruleus.
B. multicinctus snake is found in China and Taiwan while B. caeruleus snake is found in the Indian subcontinent. Of five krait species found in India, B. caeruleus is the most common one. There are about 50,000 deaths a year in India due to snakebites.
While the researchers who developed the aptamer for B. multicinctus tested it on purified alpha toxin, researchers from Translational Health Science and Technology Institute (THSTI), Faridabad , AIIMS Delhi and IIT Indore used a shorter (truncated) version of the aptamer and tested it on crude venom (which contains several proteins and enzymes besides toxin). Aside from being able to diagnose the toxin contained in B. caeruleus venom, the truncated aptamer showed greater affinity and binding to the toxin, high specificity, and ability to detect the toxin even when very little of the venom was present. The results were published in the journal Scientific Reports.
The current diagnostic approach is to use symptoms associated with snake bites and blood coagulation tests, both of which have shortcomings. Even the antibody-based immunoassays have certain challenges. In the absence of an accurate diagnostic test for snakebites, the current approach is to use large quantity of antivenom that is effective against cobra, krait, Russell’s viper and saw-scaled viper venom. But the use of polyvalent antivenom has an inherent problem — many patients suffer from mild to severe adverse reactions.
Developing tests that can accurately diagnose the toxin of a snake species will facilitate the use of monovalent antivenom for treatment, thus reducing the possibility of adverse reactions.
The research team led by Dr. Tarun Kumar Sharma from THSTI first compared the protein sequence of the alpha toxin present in B. multicinctus venom with other krait snake species. There was close similarity between the protein sequences of B. multicinctus and B. caeruleus toxins.
Based on the aptamer sequence, the researchers predicted the structure of the aptamer using online tools. The structure information was used for making two truncated versions of the original aptamer. “Tests confirmed that the structure and specificity of the aptamer remained intact after truncation,” says Prof. Amit Kumar from the Discipline of Biosciences and Biomedical Engineering at IIT Indore and a co-author of the paper.
Truncation improves aptamer properties
The ability of the aptamer to bind to the toxin present in the crude venom was next tested. While one truncated aptamer showed less binding to the toxin, the other with 26 nucleotides showed better binding than even the parent aptamer (with 40 nucleotides).
“The truncation improved the specific interaction between the aptamer and the target by about six-fold and this led to better binding,” says Abhijeet Dhiman from AIIMS and one of the first authors of the paper.
“We were able to identify the minimum sequences that were responsible for aptamer binding. Reducing the size of the aptamer used will help in reducing the cost of the diagnostic assay,” says Dr. Sharma.
The sensitivity of the truncated aptamer also increased. “The original aptamer could detect the venom only when 8 nanogram was available but the truncated version could detect the toxin when 2 nanogram of the venom was present,” says Anjali Anand from THSTI and the other first author of the paper.
Snakes of the same species from different places have inherent variability in venom. So after testing the binding ability of the aptamer to B. caeruleus krait from West Bengal, the researchers tested it on venom from Maharashtra and Uttar Pradesh. “Even with geographical variation in the venom, the binding of the aptamer to the alpha toxin was unaffected,” says Dr. Sharma.
Developing aptamers inhouse
“This is a proof-of-concept study to understand whether aptamers can be used for detecting the toxin present in crude venom,” says Dr. Sharma. “We have now developed an aptamer with different nucleotide sequences for detecting B. caeruleus krait venom. The aptamer we developed shows better binding than the existing one.” The team is in the process of testing other characteristics of their aptamer.
The researchers are also developing aptamers for detecting the venom of other poisonous snakes such as cobra, Russell’s viper and saw-scaled vipers.