Streamlining RNA Aptamer Selection via Unique Molecular Identifiers and High-Throughput Sequencing

• Published in: Analytical chemistry (Washington) 2024-10, Vol.96 (42), p.16686-16694
• Main Authors: Zhang, Dengwei, Liu, Yuqing, Huang, Huidong, Fu, Ting, Bing, Tao, Wu, Xiaoqiu, Tan, Weihong
• Format: Article
• Language: English
• Subjects: Aptamers; Chemical modification; Colon cancer; Drug delivery; Gene sequencing; Medical research; Molecular chains; Myoblasts; Next-generation sequencing; Polymerase chain reaction; Precision medicine; Ribonucleic acid; RNA; RNA modification; Target recognition
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.4c02984

Aptamers are valuable tools for applications such as cell imaging, drug delivery, and therapeutics. RNA aptamers, in particular, exhibit complex structural diversity and flexibility, affording higher affinity and specificity, broader target recognition, and easier chemical modification compared with DNA aptamers. However, traditional selection methods for RNA aptamers are time-consuming and involve numerous rounds of screening, thus limiting their widespread application. To overcome this challenge, we propose an efficient truncated selection approach termed ID-SELEX. This method incorporates a molecular identification marker whereby each template is labeled with a unique molecular identifier, or UMI. Such incorporation helps mitigate biases introduced by multiple polymerase chain reaction (PCR) amplification during high-throughput sequencing, ensuring accurate identification of aptamer candidates. Utilizing ID-SELEX, we successfully identified a panel of high-quality aptamers targeting the human colon cancer cell line HCT-8 in just 2 rounds of selection. Furthermore, we demonstrated the versatility of this strategy by selecting 6 RNA aptamers targeting mouse myoblast cell line C2C12 with only one round of selection. In summary, RNA aptamer selection based on ID-SELEX utilizes high-throughput sequencing and UMI labeling to enable the rapid screening of RNA aptamers across human and murine cell lines. As such, ID-SELEX has the potential to facilitate RNA aptamer discovery, providing a novel molecular tool for biomedical research, clinical applications, and precision medicine.