Although DNA aptamers can show comparable affinity to antibodies and have the advantage of having high batch-to-batch consistency, they often suffer from unsatisfied specificity for complex samples. The limited library size used for aptamer in vitro isolation (SELEX) has been recognized as one of the major reasons. Programmed cell death-ligand 1 (PD-L1) is both a key protein in cancer diagnostics and also immunotherapy. We report here a DNA aptamer that highly specifically binds PD-L1 expressed on the surface of various cancer cells and multiple types of tissue sections. The aptamers were selected from a DNA library containing a type II restriction endonuclease Alu I recognition site in the middle of the 40-nt random sequences, against recombinant PD-L1 rather than the whole cell or tissue section. The library enrichment was achieved by Alu I mediated-SELEX, named as REase-SELEX, in which Alu I cut off the non-binders at the recognition site and, more importantly, induced library mutations to substantially increase the library diversity. 8–60, a representative aptamer with high affinity (K_D = 1.4 nM determined by SPR) successfully detected four types of cancer cells with PD-L1 expression levels from low to high by flow cytometry, normal human tonsil (gold standard for PD-L1 antibody evaluation), clinical non-small cell lung cancer (high PD-L1 expression level), and malignant melanoma (low PD-L1 expression level) tissue sections by fluorescence microscopy imaging, showing unprecedented high specificity. The results demonstrate that 8–60 is an advanced probe for PD-L1 cancer diagnostics and mutations in SELEX greatly favor aptamer specificity.

尽管 DNA 适配体可以显示出与抗体相当的亲和力，并且具有批次间一致性高的优势，但它们通常对复杂样品的特异性不满意。用于适体体外分离 (SELEX) 的有限库大小已被认为是主要原因之一。程序性细胞死亡配体 1 (PD-L1) 是癌症诊断和免疫治疗中的关键蛋白质。我们在这里报告了一种 DNA 适体，它高度特异性地结合在各种癌细胞和多种类型的组织切片表面上表达的 PD-L1。适配体选自 DNA 文库，该文库在 40 nt 随机序列的中间含有 II 型限制性内切核酸酶 Alu I 识别位点，针对重组 PD-L1 而不是整个细胞或组织切片。文库富集是通过Alu I介导的-SELEX实现的，命名为REase-SELEX，其中Alu I切断识别位点的非结合物，更重要的是诱导文库突变，大幅增加文库多样性。8-60，具有高亲和力的代表性适体（K_D  = 1.4 nM SPR测定）成功通过流式细胞术检测出四种PD-L1表达水平由低到高的癌细胞、正常人扁桃体（PD-L1抗体评估的金标准）、临床非小细胞肺癌（高 PD-L1 表达水平）和恶性黑色素瘤（低 PD-L1 表达水平）组织切片通过荧光显微镜成像，显示出前所未有的高特异性。结果表明，8-60 是一种用于 PD-L1 癌症诊断的先进探针，SELEX 中的突变非常有利于适体特异性。