Selection Of DNA Aptamers Against Human Recombinant LRP5 Protein

Background:Malignant tumor are a common problem in the world and the first cause of death.Malignant tumors develop rapidly and easily to metastasize,which making a difficult in the treatment.The study found that the dysregulation of the Wnt signaling pathway leads to the occurrence of many kinds of tumors in human.The conduction of Wnt signaling pathway is divided intoβ-catenin-dependent classical pathway andβ-catenin-independent non-canonical pathway.Wnt protein specifically binds to FZD family proteins in the classical pathway,and induces intracellular signals under the regulation of LRP5/LRP6receptors.The transduction process regulates multiple genetic programs that play a role in cell proliferation,migration,angiogenesis,survival,and metabolism.These studies show that LRP5 and LRP6 can regulate tumor promoter/suppressor genes and thus influnce the generation and development of tumor.The study of this signaling pathway can help us to explore the mechanisms of tumor development and metastasis which can inhibit the growth and development of tumors for the purpose of treating tumors..Nucleic acid aptamers is a single-stranded DNA/RNA sequences that specifically bind to target molecules.They have many advantages such as simple synthesis,no immunogenicity,and low molecular weight.Therefore,they are also called"chemical antibodies."Nucleic acid aptamers have been widely used in many fields of biomedicine due to it’s unique advantages,especially in the development of disease diagnosis and treatment.There is currently an aptamer drug approved for clinical named-Macugen.It can specifically bind to VEGF and block its downstream signaling pathway for the purpose of treating age-related macular degeneration.At present,the aptamer screening for LRP5protein has not been reported yet.Selection of LRP5 aptamer may help us to study the tumorigenesis and metastasis mechanism and develop new drugs that are beneficial to tumor therapy.Objective:To select a high-affinity,high-specificity single-stranded DNA aptamers that bind to LRP5 protein using systematic evolution of ligands by exponential enrichment(SELEX)technique.The aptamers were sequence optimized to maintain a good affinity and specificity,and provide a new idea for the study of tumor development and tumor therapy.Methods:LRP5 protein with his-tag was interacted with nickel magnetic beads,and then LRP5 protein was immobilized on nickel magnetic beads.By SELEX technology,an 81 bp single-stranded oligonucleotide library was enriched with LRP5-beads as a target.The candidate aptamer families was obtained by cloning and sequencing.The secondary structure of the aptamer was predicted and analyzed by NUPACK software.The equilibrium dissociation constant(K_d value)and specificity of the aptamers were measured by flow cytometry.The aptamers with good property were selected for sequence optimization and flow cytometry was used to examine whether the optimized sequences had good specificity and affinity.Results:1.In this study,3 aptamers that specifically bind to LRP5 protein were successfully obtained by magnetic bead-dependent SELEX technology,after 12 rounds of selection in vitro,named L-apt1,L-apt2,and L-apt3.2.These three aptamers have good target protein binding capacity and high specificity.L-apt1 has the lowest K_d value,only 24.14±18.29 n M.3.The optimized sequence L-apt1c also has strong specificity and affinity which can be used for subsequent studies.Conclusion:This study successfully developed a new aptamer that binds human LRP-5 protein with high affinity and high specificity.To provide a new idea for the study of the development,metastasis mechanism of malignant tumors and the treatment of malignant tumors.It provides a new idea for studying the mechanism of tumor occurrence and metastasis,as well as treatment of malignant tumors.