Due to their high target binding affinity and specificity [1], low production cost and ease of modification, aptamers have been widely used as biorecognition elements in detecting drug residues, monitoring environmental pollutants and diagnosing clinical markers [2]. Moreover, aptamers can also be used as targeted agents for antibacterial, antiviral and cancer treatment [3].
Although much progress has been made in aptamer applications, there are still some bottlenecks that hinder the further utilization of aptamers. One prominent bottleneck is the stability of aptamers, especially in practical applications. The low stability and short half-life of aptamers can result in their rapid clearance rate in vivo [4], consequently leading to shortened durations for aptamer-assisted targeted therapy and imaging window time [5]. Therefore, it is urgently necessary to enhance the robustness (i.e., stability) of aptamers to bolster their applicability and effectiveness.
At present, chemical modification is the main strategy to enhance aptamer stability [6]. For example, imidazolium coordinated thymidine has been employed to modify the aptamer towards L-arginine amide to improve its detection stability [7]. However, this modification strategy would introduce extra labor and cost [8], and some aptamers may not be easily amenable to chemical modifications [9]. Additionally, chemical modifications have the potential to alter the conformation of the aptamer, thereby reducing the degree of conformational change in the process of recognizing and binding to the target substance. Consequently, this limits the sensitivity of the detection system to a certain extent [10]. Therefore, it is imperative to explore alternative strategies for enhancing aptamer stability [11].
Traditionally, aptamers were screened by the systematic evolution of ligands by exponential enrichment (SELEX) strategy, capture SELEX or other derivative strategies [12, 13]. Due to its high selectivity, sensitivity and diversity (Table S1), capture-SELEX finds extensive applications in biomedical research, including drug discovery, tumor marker identification, and biosensor development [14]. However, these approaches typically employ ordinary buffers as the screening system [15], neglecting the inclusion of real samples that the aptamers would encounter in their intended applications. It is crucial to acknowledge that the composition, and physical and chemical factors contained in complex samples would seriously affect the stability of aptamers [16], thereby discounting their detection performance. In our previous work, we introduced a real milk sample assisted SELEX strategy for selection of specific aptamer towards sarafloxacin (SAR) [17]. In this strategy, the real sample with which the aptamer would interact in real-work scenarios was employed during the screening process. This approach allowed the aptamer to undergo pre-adaptation to the complexities of the authentic sample in advance. Such pre-adaptation to real sample would confer aptamer resistance to adverse factors in the real sample. Here, to improve the aptamer stability, a real sample assisted capture-SELEX strategy illustrated by selection of specific aptamer towards PD-L1 is proposed (Fig. 1).
PD-L1 (Programmed death ligand-1), which is a member of B7 family with negative immune regulation effect [18], can specifically recognize and bind to PD-1 on immune cells. PD-1/PD-L1 pathway is an important immune checkpoint, which can inhibit the activation of T cells and help tumor cells to realize immune escape [19]. PD-L1 aptamer has been used for diagnosis and treatment of tumors, but its stability has not been considered [20]. PD-L1 aptamer with high stability would facilitate improving the efficiency of cancer diagnosis, imaging and targeted immunotherapy.
Here, a real sample assisted capture-SELEX strategy was proposed to screen aptamer towards PD-L1 from random sequence library. Based on such selection strategy, the aptamer Apt-S1 with higher PD-L1 binding affinity was obtained as compared to the aptamer Apt-A2 which was screened by the traditional capture-SELEX approach. More importantly, Apt-S1 suggested higher stability in human serum superior to that of Apt-A2. Meanwhile, defatted serum and deproteinized serum were respectively used to investigate the potential reasons for improved stability of aptamer screened by this selection strategy.