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Rapid Capture and Nondestructive Release of Extracellular Vesicles Using CD63 Aptamer

Hits:366   Date: 5/25/2023
ECALBIO company develop DNA aptamer -CD63 aptamer, is used in Rapid Capture and Nondestructive Release of Extracellular Vesicles Using Aptamer-Based Magnetic Isolation studied by School of Pharmaceutical Sciences, Zhengzhou University.
 
To address this issue, they designed a DNA aptamer-based system that enabled rapid capture and nondestructive release of EVs in 90 min with similar isolation efficiency to ultracentrifugation (around 78%). Moreover, because we designed a DNA structure-switch process to release the exosomes, the isolated EVs maintained high bioactivity in cell-uptake assay and wound-healing assays. Using this method, we can isolate EVs from clinical samples and found that the amount of MUC1 positive EVs in breast cancer patient plasma sample is significantly higher than that in healthy donors. This DNA aptamer-based magnetic isolation strategy can be potentially applied for the biofunction study of EVs and EV-based point-of-care clinical tests.
 
The specificity of CD63 aptamer was also tested. Specifically, the CD63 aptamer and random sequences were modified on glass slides, and the fluorescent labeled exosomes were incubated with the two kinds of surface and then imaged using confocal microscopy. According to the fluorescent image and exosome number quantification , the amount of exosomes captured by CD63 aptamer is significantly higher than those captured in the random sequence and PBS groups, indicating CD63 aptamer has good capture efficiency and specificity.
 
In summary, they developed a DNA aptamer-based magnetic isolation method for rapid capture and nondestructive release of EVs from cell culture medium and human plasma samples.
 
Using DNA aptamer as a recognition and capture agent, we developed this AMI method to achieve EVs with high recovery efficiency (78%) and fewer protein impurities in less than 90 min. Compared with previously developed EV isolation process, we can use complementary DNA sequences to release captured EVs from magnetic beads without any disturbance to the EV structure or surface protein markers. The bioactivity of isolated EVs was tested using cell-uptake assay and wound healing assay, which showed high potential for downstream biofunction studies. Further, AMI was used for isolating EVs from human plasma samples and showed good reproducibility. This rapid and nondestructive EV isolation method can be potentially applied for studying the biofunction of EVs or profiling the surface markers of EVs for clinical translation of diagnosis and prognosis.
 
Some methods of CD63 Aptamer use:
Specificity of CD63 Aptamer. The CD63 aptamers(5′-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TAT TTT TTT TTT TTT TTT-3′-biotin) and random sequence (5′- TGT GCG GCG AAA TAT TAT AGC TAC CGC AAT TAC TTT TTT TTT TTT TTT-3′-biotin) were synthesized by Shanghai Sangon Co., Ltd. and purified by HPLC. The experiment was conducted in small wells with a diameter of 0.5 cm, and the bottom of each well was streptavidin-modified. The exosomes form MCF-7 cells were collected and incubated with 5 μM Dil for 10 min at room temperature. The concentration of MCF-7 exosomes was counted to be 1.4 × 107 particles/mL using NTA. The collected exosomes were separated into two groups and incubated with CD63 aptamer and random sequence, respectively, then washed with PBS three times. Then, the samples were imaged using confocal microscopy. The amount of exosomes captured by CD63 aptamer and random sequence were counted by fluorescent imaging to evaluate the specificity of CD63 aptamer.
 
Aptamer-Based EV Isolation. CD63 aptamer (5′-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TAT TTT TTT TTT TTT TTT-3′-biotin) and MUC1 aptamer (5′-biotin-TTTTTTTTTT GCA GTT GAT CCT TTG GAT ACC CTG G-3′) were purchased from Sangon Biotech. EV capture steps were as follows: First, biotin labeled CD63 aptamers were used to identify and bind to EVs in a 500 μL sample. Then, the streptavidin-conjugated MBs (Invitrogen) were added to capture the aptamer labeled EVs. For capture optimization, we studied the effect of aptamer concentrations, incubation time, and MB concentration by evaluating the capture efficiency. Capture efficiency was measured by NTA.
 
Release of Captured EVs. Complementary DNA sequence (5′- GTG GGG TGG AGC GAG GGC ACT GTG ATT ACG ATA AAA AAA AAA AAA AAA-3′ and 5′-CCA GGG TAT CCA AAG GAT CAA CTG CAA AAA AAA AA-3′) was used to open the aptamer structure and release EVs from MBs. The EV release process is as follows: MBs with EVs were washed 3 times with PBS and then redistributed in 500 μL of PBS. Complementary DNA sequences were then added to the redistributed samples, which were incubated at 37 °C for 15 min. After magnetic separation, the release efficiency was calculated by measuring EV concentration of the supernatant using NTA. Isolation efficiency = amount of released EVs/amount of total EVs × 100%.
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