增殖诱导配体蛋白的核酸适配体筛选与特异性研究

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

摘要/Abstract

摘要：

获得TNF 家族增殖诱导配体（a proliferation inducing ligand,APRIL）的核酸适配体,并考察其亲和特异性。基于磁珠-SELEX技术筛选APRIL适配体,高通量测序获得特异性结合APRIL的序列,在线软件预测其二级结构,应用斑点印迹、酶联寡核苷酸吸附实验和qPCR法测定所获适配体的亲和力和特异性。经过7轮的筛选获得核酸适配体Apt10和Apt16,预测其二级结构均具有茎环结构,斑点印迹和酶联寡核苷酸吸附实验检测结果表明,2条核酸适配体均可特异性识别APRIL蛋白,酶联寡核苷酸吸附实验和qPCR检测Apt10和Apt16的解离常数均在nmol/L范围内。成功筛选获得能特异性结合APRIL的高亲和力DNA适配体。

关键词: 增殖诱导配体, 核酸适配体, 指数富集配体系统进化, 高通量测序

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

郑芳芳, 林俊生. 增殖诱导配体蛋白的核酸适配体筛选与特异性研究[J]. 生物技术通报, 2021, 37(10): 196-202.

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.

图/表 9

参考文献 21

[1]	Hahne M, Kataoka T, Schrter M, et al. APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth[J]. Journal of Experimental Medicine, 1998, 188(6):1185-1190. doi: 10.1084/jem.188.6.1185 URL
[2]	Rennert P, Schneider P, Cachero TG, et al. A soluble form of B cell maturation antigen, a receptor for the tumor necrosis factor family member APRIL, inhibits tumor cell growth[J]. Journal of Experimental Medicine, 2000, 192(11):1677-1683. doi: 10.1084/jem.192.11.1677 URL
[3]	潘超, 等. APRIL及PGR检测指标在胃炎, 胃癌中的诊断价值分析[J]. 胃肠病学和肝病学杂志, 2019, 28(6):606-609.
	Pan C, et al. Analysis of the diagnostic value of APRIL and PGR in gastritis and gastric cancer[J]. Chinese Journal of Gastroenterology and Hepatology, 2019, 28(6):606-609.
[4]	Gao Q, Li Q Xue Z, et al. In vitro and in vivo evaluation of a humanized anti-APRIL antibody[J]. Current Molecular Medicine, 2013, 13(3):464-465. pmid: 23331019
[5]	Ashkenazi AJ, Dodge KH, Grewal I, et al. Anti-APRIL monoclonal antibody and its use for the treatment of an immune related disease or cancer:EP 1666052 B1[P], 2011-06-08.
[6]	Guadagnoli M, Kimberley FC, et al. Development and characterization of APRIL antagonistic monoclonal anti-bodies for treatment of B-cell lymphomas[J]. Blood, 201, 117(25):6856-6865.
[7]	Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands[J]. Nature, 1990, 346:818-822. doi: 10.1038/346818a0 URL
[8]	Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNA polymerase[J]. Science, 1990, 249:505-510. pmid: 2200121
[9]	Groff K, Brown J, Clippinger AJ. Modern affinity reagents:Recombinant antibodies and aptamers[J]. Biotechnology Advances, 2015, 33(8):1787-1798. doi: 10.1016/j.biotechadv.2015.10.004 URL
[10]	Mercier MC, Dontenwill M, Choulier L. Selection of nucleic acid aptamers targeting tumor cell-surface protein biomarkers[J]. Cancers, 2017, 9(69):1-33. doi: 10.3390/cancers9010001 URL
[11]	Gaudin V. The growing interest in development of innovative optical aptasensors for the detection of antimicrobial residues in food products[J]. Biosensors, 2020, 10(3):21-29. doi: 10.3390/bios10030021 URL
[12]	王子健, 陈尔凝, 杨歌, 等. 小分子靶标的核酸适配体筛选研究进展[J]. 分析化学, 2020, 48(5):24-43.
	Wang ZJ, Chen EN, Yang G, et al. Research advances of aptamers selection for small molecule targets[J]. Chinese Journal of Analytical Chemistry, 2020, 48(5):24-43.
[13]	Dou H, Yan Z, Zhang M, et al. APRIL promotes non-small cell lung cancer growth and metastasis by targeting ERK1/2 signaling[J]. Oncotarget, 2017, 8(65):109289-109300. doi: 10.18632/oncotarget.v8i65 URL
[14]	Lokshin A, et al. BAFF and APRIL from ActivinA-treated dendritic cells upregulate the antitumor efficacy of dendritic cells in vivo[J]. Cancer Research, 2016, 76(17):4959-4969. doi: 10.1158/0008-5472.CAN-15-2668 URL
[15]	Tai Y, Lin L, et al. APRIL signaling via TACI mediates immunosuppression by T regulatory cells in multiple myeloma:therapeutic implications[J]. Leukemia, 2019, 33(2):426-438. doi: 10.1038/s41375-018-0242-6 URL
[16]	Dunn MR, et al. Analysis of aptamer discovery and technology[J]. Nature Reviews Chemistry, 2017, 1(10):76-92. doi: 10.1038/s41570-017-0076 URL
[17]	Lapa SA, Romashova KS, et al. Preparation of modified combinatorial DNA libraries via emulsion PCR with subsequent strand separation[J]. Molecular Biology, 2018, 52(6):854-864. doi: 10.1134/S0026893318060110 URL
[18]	Qiao N, Li J, et al. Speeding up in vitro discovery of structure-switching aptamers via magnetic cross-linking precipitation[J]. Analytical Chemistry, 2019, 91(21):13383-13389. doi: 10.1021/acs.analchem.9b00081 pmid: 31580650
[19]	Pengpumkiat S, Koesdjojo M, Rowley ER, et al. Rapid synjournal of a long double-stranded oligonucleotide from a single-stranded nucleotide using magnetic beads and an oligo library[J]. PLoS One, 2016, 11(3):e0149774-0149784. doi: 10.1371/journal.pone.0149774 URL
[20]	苏艺, 蒋灵丽, 林俊生. 小分子靶标与其核酸适配体亲和力的表征方法[J]. 中国生物工程杂志, 2019, 39(11):96-104.
	Su Yi, Jiang LL, Lin JS. Characterization of the affinity between low molecular weight targets and their aptamers[J]. China Biotechnology, 2019, 39(11):96-104.
[21]	Gao SX, Hu B, Zheng X, et al. Study of the binding mechanism between aptamer GO18-T-d and gonyautoxin 1/4 by molecular simulation[J]. Physical Chemistry Chemical Physics Pccp, 2016, 18(34):23458-23461. doi: 10.1039/C6CP00777E URL

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