Abstract：
The cell-SELEX method enables efficient selection of aptamers that bind whole bacterial cells. However, after selection, it is difficult to determine their binding affinities using common screening methods because of the large size of the bacteria. Here we propose a simple surface plasmon resonance imaging method (SPRi) for aptamer characterization using bacterial membrane vesicles, called nanosomes, instead of whole cells. Nanosomes were obtained from membrane fragments after mechanical cell disruption in order to preserve the external surface epitopes of the bacterium used for their production. The study was conducted on Bacillus cereus (B. cereus), a Gram-positive bacterium commonly found in soil, rice, vegetables, and dairy products. Four aptamers and one negative control were initially grafted onto a biochip. The binding of B. cereus cells and nanosomes to immobilized aptamers was then compared. The use of nanosomes instead of cells provided a 30-fold amplification of the SPRi signal, thus allowing the selection of aptamers with higher affinities. Aptamer SP15 was found to be the most sensitive and selective for B. cereus ATCC14579 nanosomes. It was then truncated into three new sequences (SP15M, SP15S1, and SP15S2) to reduce its size while preserving the binding site. Fitting the results of the SPRi signal for B. cereus nanosomes showed a similar trend for SP15 and SP15M, and a slightly higher apparent association rate constant kon for SP15S2, which is the truncation with a high probability of a G-quadruplex structure. These observations were confirmed on nanosomes from B. cereus ATCC14579 grown in milk and from the clinical strain B. cereus J066. The developed method was validated using fluorescence microscopy on whole B. cereus cells and the SP15M aptamer labeled with a rhodamine. This study showed that nanosomes can successfully mimic the bacterial membrane with great potential for facilitating the screening of specific ligands for bacteria.
摘要：
细胞-SELEX方法使得能够有效选择结合完整细菌细胞的适体。然而,选择后，由于细菌的大小很大，使用普通的筛选方法很难确定它们的结合亲和力。在这里，我们提出了一种简单的表面等离子体共振成像方法（SPRi），用于使用细菌膜囊泡进行适体表征，叫做纳米体，而不是整个细胞。从机械细胞破碎后的膜片段获得纳米体,以保留用于其生产的细菌的外表面表位。该研究是在蜡样芽孢杆菌(B.蜡质)，土壤中常见的革兰氏阳性菌,大米,蔬菜,和乳制品。最初将四个适体和一个阴性对照移植到生物芯片上。然后比较蜡状芽孢杆菌细胞和纳米体与固定化适体的结合。使用纳米体代替细胞提供了SPRi信号的30倍放大，从而允许选择具有更高亲和力的适体。发现适体SP15对蜡状芽孢杆菌ATCC14579纳米体最敏感和选择性。然后将其截断为三个新序列(SP15M，SP15S1和SP15S2）以减小其大小，同时保留结合位点。蜡状芽孢杆菌纳米体的SPRi信号拟合结果显示SP15和SP15M的趋势相似，SP15S2的表观缔合速率常数kon稍高，这是G-四链体结构的高概率截断。这些观察结果在来自在乳中生长的蜡状芽孢杆菌ATCC14579和来自临床菌株蜡状芽孢杆菌J066的纳米体上得到证实。使用荧光显微镜在整个蜡状芽孢杆菌细胞和用罗丹明标记的SP15M适体上验证所开发的方法。这项研究表明，纳米体可以成功地模拟细菌膜，具有促进细菌特异性配体筛选的巨大潜力。
