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Authors: Vignesh, R
Tirumurugaan, KG
Gopinathan, A
Title: Generation of single stranded oligonucleotides with affinity to bovine spermatozoa
Other Titles: National Symposium on Veterinary Research Priorities in Translational Animal Health, Production and Food Safety
Language: en
Type: Presentation
Pages: 1
Agrotags: Veterinary Science
Keywords: 20th Indian Veterinary Congress, Annual Conference of IAAVR, TANUVAS
Abstract: A better understanding of sperm biology and its surface characteristics is possible if studies are directed towards generating molecules with specific affinity to the surface characteristics (biochemical and structural configuration) of spermatozoa. Aptamers are novel class of molecules capable of forming specific threedimensional structures that enable them to bind to their targets with high affinity and specificity. In this study, a combinatorial library of 76 mer long (with a random core of 40 nucleotides flanked on either side with 27 known nucleotides) was used in the process of systematic evolution of ligands by exponential enrichment (SELEX) to bovine spermatozoa to enrich specific oligonucleotides with greater binding affinity. We optimized the conditions for SELEX and performed 10 rounds with the first five rounds were with unsorted spermatozoa and the remaining rounds to X-enriched spermatozoa (enrichment performed by an in-house protocol). The amplicons from the Round 3, Round 6, Round 8 and Round 10 were sequenced in Illumina platform and the processed reads (~20 million reads per sample) were analyzed with FASTAptamer bioinformatic tool to cluster the reads. We could identify 132 common clustered sequences across the rounds of SELEX analyzed. Based on the percentage of enrichment across the rounds, the sequences from the top ten common clusters from different rounds of SELEX were selected and labelled at their 5’end with 6FAM. The binding of the oligonucleotides to bovine spermatozoa were analyzed by imaging and flow cytometry. The oligonucleotides possessed different patterns of binding to bovine spermatozoa (fluorescence of the whole head, spot of fluorescence in head and mid- piece and tail) and flow cytometry revealed around 3.86% to 20.54 % binding. Among the clusters, the cluster 3 showed maximum percentage of binding (20.54%) and further SELEX rounds in being performed with X enriched spermatozoa to generate oligonucleotides with preferential binding to X/ Y spermatozoa
Description: TNV_20thMVC_PP_Feb-2020_PA21
Issue Date: 2020-02
Appears in Collections:Poster Presentations

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