Exploring aptamers for targeted enrichment of X sperm in bovine: unraveling selective potential

Figures & data

Table 1. Primers designed for selected aptamer sequence.

Aptamer library	5’TAGGGAAGAGAAGGACATATGAT (N40) TTGACTAGTACATGACCACTTGA3’ Constant Region Random Region Constant Region
Primer 1	5’TAGGGAAGAGAAGGACATATGAT3’
Primer 2	biotin-5′TCAAGTGGTCATGTACTAGTCAA3’

Table 2. Gene name, sequence and product size for X and Y specific primers.

Target	Gene	Sense sequence	Fragment size (bp)
X	MAOA	Forward CACGGCTGTTCTATGGGTCA	126
		Reverse TGCCCTGTGGTTTGTGATGT
Y	SRY	Forward ACTCCTGCAAAAGGAGCATCA	165
		Reverse CGCCTTTGTTAGCGAGAGTAAG

Figure 1. (a) Extraction and PCR based amplification of DNA aptamers by SELEX: Lane 1–5: 50 bp ladder (thermo scientific; cat#SM0371), amplified aptamers library, no template control (NTC), amplified aptamers library and NTC respectively; (b) PCR amplification product of eluted aptamers after each round of cell-SELEX.

Table 3. G Scores for the shortlisted aptamers.

Aptamer name	Position	Length	Sequence	G score
1	2	11	GGTGGCGGGGG	31
2	6	31	GGGAGGAGTAGGCGGGGGCTGGGAGCGGAGG	30
5	4	26	GGGGCAGAGGCGAGGAGGAAGAGCGG	31
10	3	35	GGGAGGTGGGGTGGGTGATGGAGGATGCGAGCGGG	47
12	15	11	GGTGGTGGCGG	31
16	4	37	GGGCGGAGTTGGGAGCAATGAGGTGAGGAAGGGTGGG	46
18	3	26	GGAGTCGTGGGGCGCGCTGGAGGGGG	28
20	1	29	GGGCAGTGGTGCAGTGGGGGAGCAGGCGGGGCCAGGGGG	56
22	11	23	GGGAGGGTCGGGATATAGTTGGG	55
23	25	13	GGTGGACCAGGGG	27
26	3	29	GGTTGCGTGGGGTGCGACGGGAGAAGAGG	31
28	1	13	GGCGGTGTGGGGG	29
29a	14	23	GGGACGGGGGCGGGAAGGTTGGG	58
30	2	33	GGGTAGGGGGTGGGTGCGGTGGAGGCTGGTGGG	48
31	7	32	GGAGATGCGGGGCAGCAGGTTGGGTTCGTCGG	29
32	14	24	GGAGGCAGGTGAGCGGTGGATTGG	30
33	29	11	GGGGGGGGAGG	31
35	14	24	GGGATGGGAAGGCGTAGGGGTGGG	56

Figure 2. (a) 2D And (b) 3D structures for DNA aptamer 29a. The DNA aptamer nucleotide sequence was used as input for 2D and 3D structure determination using mfold server. The given structures were selected based on the minimum gibbs free energy (ΔG = −2.57).

Figure 3. (a) Fluorescence and (b) phase contrast picture of aptamer bound sperm cells in mixed semen population of bovine.

Figure 4. A Schematic representation of (a) binding of selected aptamer to sperm cells through interaction with sperm surface proteins and (b) magnetic separation of aptamer treated sperm cells bound and unbound fractions after aptamer treatment.

Figure 5. Real-time PCR analysis for determining the level of enrichment after treatment with X sperm specific aptamer 29a. # the difference between the two groups were significant at p < 0.05 and *** difference w.r.t control group were significant at p < 0.05.