Generation of single stranded oligonucleotides with affinity to bovine spermatozoa
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Date
2020-02
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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
Keywords
Veterinary Science