A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.
Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy et al. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133 nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application.
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, No. 800, Xiangyin Rd., Shanghai 200433, People's Republic of China.
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X
Zheng
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, No. 800, Xiangyin Rd., Shanghai 200433, People's Republic of China; Center of Marine Biolical Medicine, College of Marine Military Medicine, Second Military Medical University, No. 800, Xiangyin Rd., Shanghai 200433, People's Republic of China.
B
B
Hu
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, No. 800, Xiangyin Rd., Shanghai 200433, People's Republic of China.
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SX
Gao
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, No. 800, Xiangyin Rd., Shanghai 200433, People's Republic of China; Center of Marine Biolical Medicine, College of Marine Military Medicine, Second Military Medical University, No. 800, Xiangyin Rd., Shanghai 200433, People's Republic of China.
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DJ
Liu
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, No. 800, Xiangyin Rd., Shanghai 200433, People's Republic of China.
M J
MJ
Sun
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, No. 800, Xiangyin Rd., Shanghai 200433, People's Republic of China; Center of Marine Biolical Medicine, College of Marine Military Medicine, Second Military Medical University, No. 800, Xiangyin Rd., Shanghai 200433, People's Republic of China.
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BH
Jiao
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, No. 800, Xiangyin Rd., Shanghai 200433, People's Republic of China. Electronic address: wsh928@hotmail.com.
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LH
Wang
D000818:Animals; D052157:Aptamers, Nucleotide / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005240:Feasibility Studies; D005260:Female; D051379:Mice; D008813:Mice, Inbred ICR; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012530:Saxitoxin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000633:toxicity
D052157:Aptamers, Nucleotide; D012530:Saxitoxin
Aptamer; G-quadruplex; Inhibitory activity; Mutagenesis; Saxitoxin; Truncation