We
report here a modified aptamer selection method, magnetic cross-linking
precipitation (MCP)-SELEX, for highly efficient library enrichment
and aptamer isolation. MCP-SELEX isolates bound aptamers via highly
efficient chemical cross-linking between amino groups of target proteins
and activated carboxylic acid groups on magnetic beads (>90% coupling
efficiency). Importantly, MCP-SELEX avoids surface interferences in
conventional target-fixed methods and substantially minimizes nonspecific
binding. The enrichment efficiencies of MCP-SELEX for various proteins
(PD-L1, ubiquitin, thrombin, and HSA) were all greatly higher than
those of the conventional target-bound magnetic bead based-SELEX (MB-SELEX).
Antithrombin aptamer with KD of 33 nM was successfully
isolated by four rounds of MCP-SELEX. MCP-SELEX also enabled the efficient
aptamer isolation by coupling with MB-SELEX or falling-off-SELEX.
We identified structure-switching aptamers (SSAs) that specifically
bind to HSA with low nanomolar dissociation constant via three rounds
of MCP-SELEX and 1 round of falling-off-SELEX. Our HSA SSAs also have
∼3-fold higher specificity against streptavidin relative to
thrombin SSAs discovered through falling-off-SELEX only. The enriched
library has ∼78-fold higher signal-to-noise ratio (the number
of DNAs eluted by 50 nM HSA divided by the number of DNAs self-dissociated
in blank buffer) than that obtained by 4 rounds of direct falling-off-SELEX.
We finally demonstrated the application of the selected SSA in fluorescent
detection of HSA in urine with diagnostic required sensitivity and
dynamic range. We expect that MCP-SELEX may be coupled with other
selection methods to substantially accelerate aptamer discovery.
PD-1/PD-L1 is an important pathway in immunotherapy and the high PD-L1 expression level in tumor tissues is an essential prerequisite for PD-1/PD-L1 blocking-based therapy. The PD-L1 expression level in tumor...
Orthopedic implants have a high failure rate due to insufficient interfacial osseointegration, especially under osteoporotic conditions. Type H vessels are CD31 + EMCN + capillaries with crucial roles in mediating new bone formation, but their abundance in osteoporotic fracture site is highly limited. Herein, we report a nanoengineered composite coating to improve the in situ osseointegration of a Ti implant for osteoporotic fracture repair, which is realized through inhibiting the stimulator of interferon genes (STING) in endothelial cells (ECs) to stimulate type H vessel formation. Autonomously catalytic DNAzyme-ZnO nanoflowers (DNFzns) were prepared through rolling circle amplification (RCA) of STING mRNAdegrading DNAzymes, which were then integrated on the Ti surface and further sequentially complexed with thioketal-bridged polydopamine and naringenin (Ti/ DNFzn/PDA-Nar). ECs and mesenchymal stem cells (MSCs) can be recruited to the implant surface by galvanotaxis, accounting for the negative charges of DNFzn/PDA-Nar, subsequently released Nar under reactive oxygen species (ROS) stimulation to upregulate endothelial nitric oxide synthase (eNOS) in recruited ECs, leading to enhanced local angiogenesis. Meanwhile, the coordinately released DNFzns would abolish STING expression in ECs to transform the newly formed vessels into Type H vessels, thus substantially promoting the osseointegration of Ti implants. This study provides application prospects for improving implant osteointegration for osteoporotic fracture treatment.
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