Selection and Specificity of Nucleic Acid Aptamers for a Proliferation Inducing Ligand

doi:10.13560/j.cnki.biotech.bull.1985.2021-0069

Abstract

Abstract:

It is aimed to obtain aptamers against a proliferation inducing ligand（APRIL）of TNF family and to investigate their affinities and specificities. Based on systematic evolution of ligands by exponential enrichment（SELEX）,magnetic bead coupling target method was used to select APRIL protein-specific aptamers. High-throughput sequencing technology was applied to have candidate aptamers sequenced. Online software was to predict their secondary structures. Dot blotting,enzyme-linked oligonucleotide assay（ELONA）and qPCR methods were to characterize their target-binding affinities and specificities. Aptamers Apt10 and Apt16 were obtained after seven rounds of selection. Their predicted secondary structures all showed a stem-loop structure. Dot blotting and ELONA showed that the two aptamers specifically recognized APRIL protein. Results of the ELONA and qPCR indicated that their dissociation constants both were at nmol/L level. Two DNA aptamer sequences against APRIL with high affinity and specificity were successfully prepared.

Key words: a proliferation inducing ligand, nucleic acid aptamer, systematic evolution of ligands by exponential enrichment, high-throughput sequencing

ZHENG Fang-fang, LIN Jun-sheng. Selection and Specificity of Nucleic Acid Aptamers for a Proliferation Inducing Ligand[J]. Biotechnology Bulletin, 2021, 37(10): 196-202.

Figures/Tables 9

References 21

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Item	Sequence of random region（5'-3'）	Round 4/%	Round 5/%	Round 6/%	Round 7/%	dG/ （kcal·mol^-1）	Stem-loop structure	G/%
Apt 1	GGTTGGGTTGGGGGGGTACTCTGAACTCGCATAGCCGGTG	0.7	1.4	3.8	4.7	-8.9	4	45.0
Apt 2	AAGAGGCGGGCGGGTGGGGCTGACCTCGCACTTGGTTCTC	0.3	1.5	2.5	5.2	-8.2	5	42.5
Apt 3	TGGTGGGAGGTATACGGGTGGTGGGTGGGGGTGTTGATGG	0.1	3.1	3.3	3.4	-7.5	5	60.0
Apt 5	GATCCTCGCGTAGGCTCGGGGTGAGGTGGGTGGGTTCGTG	0.6	2.2	2.9	3.7	-10.0	4	50.0
Apt 7	GGTCGGGGTGGGTGGGCGACCTCGCATAAGTTAGGCAGTT	0.2	2.7	3.0	3.9	-8.7	4	45.0
Apt 10	ACTGGGGGGGGTCGGGTGGGTGTCTCGGACCTCGCATCA	0.5	2.3	3.1	3.8	-8.8	5	46.2
Apt 14	TGTTGGGTTGGGGGGGTACTCTGAACTCGCATAGCCGGTG	0.1	1.4	4.2	4.2	-9.0	3	42.5
Apt 16	GGTCGGGTGGGTGGGCGACCTCGCATAGCGATTGATGATC	0.1	1.0	4.2	4.6	-11.5	3	42.5

Item	Sequence of random region（5'-3'）	Round 4/%	Round 5/%	Round 6/%	Round 7/%	dG/ （kcal·mol^-1）	Stem-loop structure	G/%
Apt 1	GGTTGGGTTGGGGGGGTACTCTGAACTCGCATAGCCGGTG	0.7	1.4	3.8	4.7	-8.9	4	45.0
Apt 2	AAGAGGCGGGCGGGTGGGGCTGACCTCGCACTTGGTTCTC	0.3	1.5	2.5	5.2	-8.2	5	42.5
Apt 3	TGGTGGGAGGTATACGGGTGGTGGGTGGGGGTGTTGATGG	0.1	3.1	3.3	3.4	-7.5	5	60.0
Apt 5	GATCCTCGCGTAGGCTCGGGGTGAGGTGGGTGGGTTCGTG	0.6	2.2	2.9	3.7	-10.0	4	50.0
Apt 7	GGTCGGGGTGGGTGGGCGACCTCGCATAAGTTAGGCAGTT	0.2	2.7	3.0	3.9	-8.7	4	45.0
Apt 10	ACTGGGGGGGGTCGGGTGGGTGTCTCGGACCTCGCATCA	0.5	2.3	3.1	3.8	-8.8	5	46.2
Apt 14	TGTTGGGTTGGGGGGGTACTCTGAACTCGCATAGCCGGTG	0.1	1.4	4.2	4.2	-9.0	3	42.5
Apt 16	GGTCGGGTGGGTGGGCGACCTCGCATAGCGATTGATGATC	0.1	1.0	4.2	4.6	-11.5	3	42.5