Generation Of DNA Aptamers Against Human Sperm And Preliminary Study For The Inhibitive Effect Of The Aptamers On The Sperm Motility

Although many contraceptives are available on the market with effect on reduction of the rate of unwanted pregnancy, their side effects cannot be ignored. It has been being a research hotspot for family planning services to develop new contraceptives with more safety while effectively decrease unwanted pregnancy rate.Aptamers are single-stranded oligonucleic acid molecules that can bind to a variety of target molecules with high affinity and specificity through a unique three-dimensional structure. It has a great potential to be used in the fields of basic research, disease diagnostics and treatment, because it has a lot of advantages, such as easily synthesized, small molecular size, easy to modify, wide range of target molecules,easy to remove from the blood and so on. In the present study, sperm cells were used as target cell to generate aptamers with high specificity and affinity by means of a modified Systematic Evolution of Ligands by Exponential Enrichment(cell-SELEX)technique. The effect on inhibition of sperm motility has been investigated by means of capillary tube test in vitro.The modification included combination of continually enhanced mutation and progressive selection. Mutagenesis PCR was performed for coupling DNA amplification with mutation to diversify the successive libraries. The selection conditions were getting harsher round by round to increase the recovery rate of aptamer candidates. In order to monitor the enrichment degree of anti-human sperm aptamers, we chose Dot blotting and Eastern blotting as detection means. In the end,we used the final library from the 40 th rounds for cloning and sequencing. Six of the last candidates were sequenced and confirmed as apatmers against human sperm.The candidate clones from the final library of the 40 th rounds were used as templates for PCR. The corresponding aptamers were then generated by combination of PCR and preparation of single-stranded DNA(ssDNA). The specificity of these aptamers was investigated by Dot blotting. Two aptamers, Ap-8 and Ap-9, with high specificity and affinity to sperms were determined and further verified by subsequentEastern blotting and fluorescence detection.An modified capillary tube test was designed to detect sperm motility in vitro based on the standard protocol recommended by the WHO Laboratory Manual for the Examination and Processing of Human Semen. All 20 aptamers sequences against human sperm including that generated by this study and produced by other colleagues previously were chosen for further investigation by the modified capillary tube test.The results showed that aptamer Y1623rc1, aptamer a-2Rc, aptamer a-5, aptamer a-6Rc, aptamer a-12 Rc, aptamer Ap-9 and aptamer Ap-6Rc have good ability to inhibit sperm motility.The present study has obtained six aptamers sequences with high specificity and affinity to human sperms. The effect of the aptamers on inhibition of sperm motility has been investigated. Our data provided a reference for developing new contraceptives for external application.