A Study On The Application Of Aptamer In Non-Invasive Prenatal Diagnosis

Prenatal diagnosis is the most effective way to prevent the birth of fetuses with genetic defects.Current prenatal diagnosis mainly includes invasive prenatal diagnosis(chorionic sampling,amniocentesis sampling,and umbilical cord aspiration sampling)and noninvasive prenatal diagnosis(ultrasound examination,serum screening,and cell-free fetal DNA / m RNA and fetal cells examination from maternal peripheral blood).In particular,non-invasive prenatal diagnosis based on free fetal cells from the peripheral blood of pregnant women is one of great concern.During a pregnancy,fetal cells can migrate from the fetus to the pregnant woman’s peripheral blood.Among them,fetal nucleated red blood cells(f NRBCs)in the peripheral blood of pregnant women are considered to be the most ideal materials for noninvasive prenatal diagnosis,f NRBCs in peripheral blood have a short half-life,carry complete fetal genetic information,and have specific cell markers.However,it is the key bottleneck for its clinical application that how to efficiently capture and isolate extremely rare f NRBCs from the peripheral blood of pregnant women.Aptamers are single-stranded oligonucleotides(DNA or RNA)molecules,and screened from oligonucleotide libraries by Systematic Evolution of Ligands by Exponential Enrichment(SELEX),usually less than 100 nucleotides 。And them can be folded into a unique tertiary structure,with high affinity and specific binding to target molecules.Compared with antibodies in enriching and capturing target molecules,nucleic acid aptamers have the advantages of easy immobilization and modification and good thermal stability。In this paper,nucleic acid aptamers are used as recognition ligands to achieve efficient capture and isolation of f NRBCs for noninvasive prenatal diagnosis.The main findings are as follows:(1)The nucleic acid aptamer TY7 obtained in the previous research work could specifically recognize the erythroleukemia cell line HEL,hematopoietic stem cell-induced nucleated red blood cells and f NRBCs from cord blood.Both flow cytometry and confocal results showed that TY7 could bind to HEL and nucleated red blood cells induced by hematopoietic stem cells,and the dissociation constant of binding to the erythroleukemia cell line HEL was 65.31 ± 9.3n M.Moreover,even in an extremely complicated clinical cord blood system,the aptamer TY7 can still specifically bind to f NRBCs with GPA + and CD71 +.(2)Using the nucleic acid aptamer TY7 as a ligand,the separation and capture of nucleated red blood cells can be achieved.On the one hand,the modified well plate using TY7 could specifically capture HEL cells in the binding buffer system.On the other hand,it can still specifically capture and separate HEL from the mixed system of HEL and peripheral blood mononuclear cells(PBMC)by coupling TY7 with magnetic beads,with a capture efficiency of 63.66%.Finally,it was used to capture and separate f NRBCs in clinical cord blood samples.The results showed that a large number of Hoechst + and CD71 + nucleated cells were present in the isolated cells,but the purity was lower.(3)Given that the low purity of nucleated red blood cells isolated from clinical samples by TY7,we performed cell-SELEX to obtain f NRBC-specific aptamer.During the screening process,HEL is used as positive cells and THP-1 is used as negative cells.After 22 rounds of screening,high-throughput sequencing analysis was performed on the screening products,and candidate sequences were selected to predict the secondary structure.The results showed that the nucleic acid aptamer NRBC-5 bound to HEL cells with good affinity and specificity.The dissociation constant of NRBC-5 combined with HEL cells was 153 ± 20.88 n M,but its clinical application needs further verification.The above research results indicate that nucleic acid aptamers can capture and separate f NRBCs,which lay a foundation for promoting the application of aptamers in the field of noninvasive prenatal diagnosis.