Laboratory of Precision Oral Health and Chronobiology, College of Dentistry, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, S7N 5E4, Canada; Division of Biomedical Engineering, University of Saskatchewan, 57 Campus Dr, Saskatoon, S7K 5A9, Canada; Laboratory of Oral, Head and Neck Cancer - Personalized Diagnostics and Therapeutics, Department of Surgery, College of Medicine, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, S7N 5E4, Canada.
Department of Chemistry, Faculty of Science, Carleton University, 1125 Colonel by Drive, Ottawa, Ontario, K1S 5B6, Canada.
Anal Chim Acta. 2023 Apr 22;1251:340971. doi: 10.1016/j.aca.2023.340971. Epub 2023 Feb 15.
Circadian desynchrony with the external light-dark cycle influences the rhythmic secretion of melatonin which is among the first signs of circadian rhythm sleep disorders. An accurate dim light melatonin onset (established indicator of circadian rhythm sleep disorders) measurement requires lengthy assays, and antibody affinities alterations, especially in patients with circadian rhythm disorders whose melatonin salivary levels vary significantly, making antibodies detection mostly inadequate. In contrast, aptamers with their numerous advantages (e.g., target selectivity, structural flexibility in tuning binding affinities, small size, etc.) can become preferable biorecognition molecules for salivary melatonin detection with high sensitivity and specificity. This study thoroughly characterizes the structural property and binding mechanism of a single-stranded DNA aptamer full sequence (MLT-C-1) and its truncated versions (MLT-A-2, MLT-A-4) to decipher its optimal characteristics for saliva melatonin detection. We use circular dichroism spectroscopy to determine aptamers' conformational changes under different ionic strengths and showed that aptamers display a hairpin loop structure where few base pairs in the stem play a significant role in melatonin binding and formation of aptamer stabilized structure. Through microscale thermophoresis, aptamers demonstrated a high binding affinity in saliva samples (MLT-C-1F K = 12.5 ± 1.7 nM; MLT-A-4F K = 11.2 ± 1.6 nM; MLT-A-2F K = 2.4 ± 2.8 nM; limit-of-detection achieved in pM, highest sensitivity attained for MLT-A-2F aptamer with the lowest detection limit of 1.35 pM). Our data suggest that aptamers are promising as biorecognition molecules and provide the baseline parameters for the development of an aptamer-based point-of-care diagnostic system for melatonin detection and accurate profiling of its fluctuations in saliva.
与外部明暗周期不同步会影响褪黑素的节律分泌，褪黑素是昼夜节律睡眠障碍的最初迹象之一。准确测量微弱光褪黑素起始时间（昼夜节律睡眠障碍的既定指标）需要进行冗长的检测，而且抗体亲和力会发生变化，尤其是在褪黑素唾液水平变化显著的昼夜节律障碍患者中，这使得抗体检测大多不够充分。相比之下，适体具有许多优势（例如，靶标选择性、结合亲和力的结构灵活性、体积小等），可以成为用于唾液褪黑素检测的首选生物识别分子，具有高灵敏度和特异性。本研究全面表征了单链 DNA 适体全长序列（MLT-C-1）及其截断版本（MLT-A-2、MLT-A-4）的结构特性和结合机制，以揭示其用于唾液褪黑素检测的最佳特性。我们使用圆二色光谱法确定了不同离子强度下适体的构象变化，并表明适体呈现发夹环结构，其中茎中的少数碱基对在褪黑素结合和适体稳定结构形成中起重要作用。通过微尺度热泳，适体在唾液样本中表现出高结合亲和力（MLT-C-1F K=12.5±1.7 nM；MLT-A-4F K=11.2±1.6 nM；MLT-A-2F K=2.4±2.8 nM；达到皮摩尔级别的检测限，MLT-A-2F 适体的灵敏度最高，检测限低至 1.35 pM）。我们的数据表明，适体作为生物识别分子具有很大的潜力，并为开发基于适体的即时诊断系统提供了用于检测褪黑素及其在唾液中波动的基本参数。
